KR20220166605A - Yeast strain in which all genes involved in galactose utilization are deleted and method for producing recombinant protein using the same - Google Patents
Yeast strain in which all genes involved in galactose utilization are deleted and method for producing recombinant protein using the same Download PDFInfo
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- KR20220166605A KR20220166605A KR1020210075556A KR20210075556A KR20220166605A KR 20220166605 A KR20220166605 A KR 20220166605A KR 1020210075556 A KR1020210075556 A KR 1020210075556A KR 20210075556 A KR20210075556 A KR 20210075556A KR 20220166605 A KR20220166605 A KR 20220166605A
- Authority
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- Prior art keywords
- strain
- galactose
- recombinant protein
- gene
- yeast
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- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
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Abstract
Description
본 발명은 갈락토스 이용에 관여하는 모든 유전자가 결손된 효모 균주를 사용하여 재조합 단백질을 생산하는 방법에 관한 것이다. The present invention relates to a method for producing a recombinant protein using a yeast strain lacking all genes involved in galactose utilization.
의약용, 산업용 효소 등을 대량생산하기 위해, 재조합 단백질 발현 기술이 널리 사용되고 있다. 이러한 재조합 단백질 생산에 있어서 고려해야 할 가장 중요한 요소는 적절한 숙주 세포와 발현 벡터 선정이다. In order to mass-produce pharmaceutical and industrial enzymes, recombinant protein expression technology is widely used. The most important factor to consider in the production of these recombinant proteins is the selection of appropriate host cells and expression vectors.
재조합 단백질 생산을 위한 숙주 세포로는 대장균, 효모, 동물 세포주 등이 이용되고 있다. 이들 중 효모 균주인 사카로마이세스 세레비지에 (Saccharomyces cerevisiae)는 안전성이 입증된 GRAS(Generally Regarded as Safe) 단세포 진핵생물(unicellular eukaryote)로서 단기간에 고밀도 배양이 가능하기 때문에 재조합 단백질의 대량생산이 가능하고, 봉입체를 형성하는 원핵세포 발현(prokaryotic expression) 시스템과는 달리 단백질 분비 경로가 잘 발달하였고 동물 세포와 유사한 합성 후 수식 시스템을 가지고 있기 때문에 고등동물 유래의 단백질 생산에 적합한 장점이 있어, 재조합 단백질 발현에 많이 사용되는 시스템 중 하나이다[Romanos 등, Yeast8, 423-488 (1992)].As host cells for recombinant protein production, Escherichia coli, yeast, and animal cell lines are used. Among them, Saccharomyces cerevisiae , a yeast strain, is a GRAS (Generally Regarded as Safe) unicellular eukaryote whose safety has been proven, and mass production of recombinant proteins is possible because it can be cultured at high density in a short period of time. Unlike the prokaryotic expression system that forms inclusion bodies, the protein secretion pathway is well developed and has a post-synthetic modification system similar to that of animal cells, which is advantageous for the production of proteins derived from higher animals. It is one of the systems widely used for protein expression [Romanos et al., Yeast8, 423-488 (1992)].
이러한 사카로마이세스 세레비지에 발현 벡터에서 주로 사용되는 구성적 프로모터로는 GAPDH 유전자 프로모터, TEF3 유전자 프로모터 등이 있으며, 발현 조절 프로모터로는 ADH 프로모터, 갈락토스 이용 관련 유전자 프로모터 등이 있다. 갈락토스 이용 관련 유전자는 포도당에 의해 전사가 억제되고 갈락토스 존재시 약 1000배 이상의 발현이 유도되어 구성적 프로모터보다도 강력한 발현을 유도할 수 있기 때문에 재조합 단백질의 유도 발현에 가장 많이 사용되어 왔다.Constitutive promoters mainly used in expression vectors for Saccharomyces cerevisiae include a GAPDH gene promoter and a TEF3 gene promoter, and expression control promoters include an ADH promoter and a galactose utilization-related gene promoter. Since transcription of genes related to galactose utilization is inhibited by glucose and expression is induced by about 1000 times or more in the presence of galactose, they can induce stronger expression than constitutive promoters, so they have been most frequently used for induced expression of recombinant proteins.
갈락토스 이용에 관여하는 유전자(GAL 유전자) 중 GAL2, GAL1, GAL10 및 GAL7 유전자는 배지에 포도당이 존재하거나 갈락토스가 없는 조건에서는 필요치 않기 때문에 전사 단계에서 엄격하게 조절된다. 갈락토스 존재 조건에서 효모 세포가 갈락토스를 이용하기 위해서는 갈락토스를 퍼미아제(permease, GAL2)에 의하여 세포 내로 이동시킨 후 갈락토키나아제(galactokinase, GAL1)에 의하여 갈락토스 1-인산으로 전환하고, 갈락토스 1-인산 유리딜 전이효소(galactose 1-phosphate uridyl transferase, GAL7)와 에피머라제(epimerase, GAL10)에 의하여 포도당 1-인산으로 전환하는 과정이 필요하며, 이후 해당과정을 통하여 분해시킨다. Among genes involved in galactose utilization (GAL genes), GAL2, GAL1, GAL10, and GAL7 genes are strictly regulated in the transcriptional stage because they are not required in the presence of glucose in the medium or in the absence of galactose. In order for yeast cells to use galactose in the presence of galactose, galactose is moved into cells by permease (GAL2), converted to galactose 1-phosphate by galactokinase (GAL1), and galactose 1- A process of conversion to glucose 1-phosphate by galactose 1-phosphate uridyl transferase (GAL7) and epimerase (GAL10) is required, and then it is decomposed through glycolysis.
상기 GAL 유전자의 발현은 3가지 조절 단백질, Gal4p, Gal80p 및 Gal3p에 의해 조절된다. Gal4p가 GAL 유전자의 프로모터 부위에 있는 UAS(upstream activation sequence)에 결합하여 GAL 유전자의 발현을 유도하는데, 갈락토스가 존재하지 않으면 Gal80p가 Gal4p와 결합하여 Gal4p의 기능을 저해(repression)한다. 갈락토스가 존재하는 경우에는 Gal3p가 Gal80p의 Gal4p에 대한 저해를 풀어주어 GAL 유전자가 발현된다. 반면, 포도당이 존재하는 경우 포도당을 우선적으로 사용하기 위하여 포도당이 Gal4p의 농도를 감소시켜 GAL 유전자의 전사를 저해한다. 또다른 억제인자인 Mig1p는 GAL4 URS(upstream repression sequence)에 결합하여 GAL 유전자의 전사를 저해하지만 Gal80p에 비하여 영향은 적다. Expression of the GAL gene is regulated by three regulatory proteins, Gal4p, Gal80p and Gal3p. Gal4p binds to UAS (upstream activation sequence) in the promoter region of the GAL gene to induce expression of the GAL gene. In the absence of galactose, Gal80p binds to Gal4p and represses the function of Gal4p. In the presence of galactose, Gal3p releases the inhibition of Gal80p to Gal4p, and the GAL gene is expressed. On the other hand, when glucose is present, glucose reduces the concentration of Gal4p to inhibit the transcription of the GAL gene in order to use glucose preferentially. Another repressor, Mig1p, binds to the GAL4 URS (upstream repression sequence) and inhibits the transcription of the GAL gene, but the effect is smaller than that of Gal80p.
이러한 GAL 유전자의 프로모터를 재조합 단백질 발현에 이용하기 위해서는 갈락토스가 유도물질로서 일정 농도 이상 반드시 유지되어야 하는데, 갈락토스는 탄소원으로 계속 사용되기 때문에 갈락토스의 지속적 공급이 필요하다. 그러나, 갈락토스는 포도당에 비하여 수십 배 비싸기 때문에 대량발효시 지속적으로 공급하기에는 어려움이 있다. In order to use such a GAL gene promoter for recombinant protein expression, galactose must be maintained at a certain concentration or higher as an inducer. Since galactose is continuously used as a carbon source, a continuous supply of galactose is required. However, since galactose is tens of times more expensive than glucose, it is difficult to continuously supply it during mass fermentation.
이를 해결하기 위한 방법으로, 갈락토스 대사의 두번째 과정인 갈락토키나아제를 코딩하는 GAL1 유전자를 결손시켜 갈락토스를 탄소원으로 사용할 수 없고 오로지 유도물질로만 사용하는 GAL1 결손 변이주가 개발된 바 있다[Kang HA 등, Biotechnol. Bioeng. 89, 619-629(2005)]. 상기 변이주는 적은 양의 갈락토스로 야생균주와 유사한 효과를 나타내었으나, 여전히 일정 농도 이상의 갈락토스를 필요로 하고 세포 성장이 지연되는 단점이 있었다. As a way to solve this problem, a GAL1 gene encoding galactokinase, the second process of galactose metabolism, has been deleted, and a GAL1-deficient mutant strain that cannot use galactose as a carbon source and uses it only as an inducer has been developed [Kang HA et al. Biotechnol. Bioeng. 89, 619-629 (2005)]. The mutant strain showed an effect similar to that of the wild strain with a small amount of galactose, but it still required a certain concentration or more of galactose and had a disadvantage in that cell growth was delayed.
이에, 이러한 문제점을 해결하고자 갈락토스를 사용하지 않고 GAL 유전자가 발현될 수 있는 GAL80 결손 균주가 개발되었다. Gal80p가 없으면 Gal4p를 저해할 수 없기 때문에 갈락토스가 없고 포도당만 있는 조건에서도 GAL 유전자는 Gal4p의 작용에 의하여 구성적으로 발현되었다. 또한, 해당 균주에서 GAL10 프로모터를 이용하여 재조합 단백질을 발현할 경우 갈락토스를 사용하지 않고도 GAL1 결손 균주에서 소량의 갈락토스를 사용하여 발현한 경우보다 세포 성장과 재조합 단백질 발현량이 더 높은 것이 보고되었다[Whang Jake 등, Process Biochemistry, 44, 1190-1192(2009)]. 그러나, GAL80 결손 균주는 여전히 유가식 배양으로 고농도 배양한 경우 야생 균주에 비하여 성장이 느리고, 이에 따라 발현되는 재조합 단백질 양도 감소하는 것을 확인하였다. 이러한 문제는 탄소원으로 갈락토스를 사용하지 않는 경우 GAL 유전자를 발현할 필요가 없지만 GAL80 결손 균주에서는 배지에 갈락토스가 존재하지 않는 경우에도 GAL 유전자들을 수백-수천배 과발현 상태로 유지하기 위하여 에너지를 사용하기 때문에 발생하는 것으로 추정되었다. Accordingly, in order to solve this problem, a GAL80 deficient strain capable of expressing the GAL gene without using galactose has been developed. Since Gal4p cannot be inhibited in the absence of Gal80p, the GAL gene was constitutively expressed by the action of Gal4p even in the presence of only glucose and no galactose. In addition, it has been reported that when the recombinant protein is expressed using the GAL10 promoter in the strain, cell growth and the expression of the recombinant protein are higher than when the recombinant protein is expressed using a small amount of galactose in the GAL1-deficient strain without using galactose [Whang Jake et al., Process Biochemistry, 44, 1190-1192(2009)]. However, it was confirmed that the GAL80-deficient strain still grew slower than the wild strain when cultured at high concentrations in fed-batch culture, and accordingly, the amount of recombinant protein expressed was reduced. This problem is due to the fact that GAL genes do not need to be expressed when galactose is not used as a carbon source, but in the GAL80-deficient strain, energy is used to maintain GAL genes in an overexpressed state by hundreds or thousands of times even when galactose is not present in the medium. was presumed to occur.
이러한 배경 하에서, 본 발명자들은 산업적으로 적용 가능한 재조합 단백질 생산 균주를 개발하기 위해 예의 노력한 결과, GAL80 결손 균주에서 GAL 유전자를 모두 제거한 균주(allgal)를 제작하였으며, 제작된 균주에서 GAL10 프로모터를 이용하여 재조합 단백질을 발현할 경우 균체 성장이 21-38% 증가하고 그에 따라 단백질 발현량도 12-22% 증가하는 것을 확인함으로써 본 발명을 완성하였다.Under this background, the present inventors made diligent efforts to develop industrially applicable recombinant protein production strains, and as a result, produced a strain (allgal) in which all GAL genes were removed from a GAL80-deficient strain, and recombined using the GAL10 promoter in the produced strain. The present invention was completed by confirming that cell growth increased by 21-38% when the protein was expressed, and the amount of protein expression increased by 12-22% accordingly.
본 발명의 하나의 목적은 GAL80 유전자 및 갈락토스 대사 관련 유전자가 결손된, 재조합 단백질 발현용 효모 변이주를 제공하는 것이다.One object of the present invention is to provide a yeast mutant for expression of a recombinant protein in which the GAL80 gene and the gene related to galactose metabolism are missing.
본 발명의 다른 하나의 목적은 재조합 단백질 생산 방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing a recombinant protein.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시 형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.
본 발명의 하나의 양태는 GAL80 유전자 및 갈락토스 대사 관련 유전자가 결손된, 재조합 단백질 발현용 효모 변이주를 제공한다.One aspect of the present invention provides a yeast mutant for expression of a recombinant protein in which the GAL80 gene and the gene related to galactose metabolism are missing.
구체적으로, 상기 갈락토스 대사 관련 유전자는 GAL1, GAL10, GAL7 및 GAL2 유전자로 이루어지는 군으로부터 선택되는 어느 하나 이상인 것일 수 있다.Specifically, the gene related to galactose metabolism may be at least one selected from the group consisting of GAL1, GAL10, GAL7 and GAL2 genes.
본 발명에서 용어, "GAL"은 갈락토스 이용에 관여하는 유전자를 총칭하는 것일 수 있다.In the present invention, the term "GAL" may refer to genes involved in galactose utilization.
본 발명에서 용어, "GAL80"은 갈락토스 이용에 관여하는 유전자(GAL 유전자) 중 하나로, 또 다른 갈락토스 이용에 관여하는 유전자인 GAL4 이량체에 결합하여 GAL4가 UAS(upstream activation sequence)에 결합하더라도 전사를 활성화할 수 없도록 하는, GAL4 전사 활성제(transcriptional activator)의 음성 조절자(negative regulator)를 의미한다.In the present invention, the term "GAL80" is one of the genes (GAL gene) involved in the use of galactose, which binds to GAL4 dimer, another gene involved in the use of galactose, and activates transcription even when GAL4 binds to UAS (upstream activation sequence). It refers to a negative regulator of the GAL4 transcriptional activator, which renders it inactivated.
본 발명에서 용어, "GAL1"은 갈락토스 이용에 관여하는 유전자 중 하나로, 갈락토키나아제(galactokinase)를 코딩하는 유전자를 의미한다. 상기 갈락토키나아제는 갈락토스 대사의 첫 번째 단계에서 세포질에서 알파-D-갈락토스(alpha-D-galactose)를 알파-D-갈락토스-1-인산(alpha-D-galactose-1-phosphate)로 인산화하는 효소이다.As used herein, the term "GAL1" is one of the genes involved in galactose utilization, and refers to a gene encoding galactokinase. The galactokinase phosphorylates alpha-D-galactose to alpha-D-galactose-1-phosphate in the cytosol in the first step of galactose metabolism. is an enzyme
본 발명에서 용어, "GAL10"은 갈락토스 이용에 관여하는 유전자 중 하나로, UDP(uracil-diphosphate)-글루코스-4-에피머라제 및 알도스 1-에피머라제(UDP-glucose-4-epimerase and aldose 1-epimerase) 활성을 동시에 가지는 유전자를 코딩하는 유전자를 의미한다. 상기 이중 기능성 UDP(uracil-diphosphate)-글루코스-4-에피머라제 및 알도스 1-에피머라제는 갈락토스 대사에서 갈락토스 대사에서 UDP-갈락토스와 UDP-D-글루코스의 상호 전환을 촉매하고, 알파-D-글루코스 또는 알파-D-갈락토스의 베타-아노머로(beta-anomers)의 전환을 촉매하는 효소이다.In the present invention, the term "GAL10" is one of the genes involved in galactose utilization, UDP (uracil-diphosphate) -glucose-4-epimerase and aldose 1-epimerase (UDP-glucose-4-epimerase and aldose 1-epimerase) means a gene that encodes a gene that simultaneously has activity. The bifunctional UDP (uracil-diphosphate)-glucose-4-epimerase and aldose 1-epimerase catalyze the interconversion of UDP-galactose and UDP-D-glucose in galactose metabolism, and alpha- It is an enzyme that catalyzes the conversion of D-glucose or alpha-D-galactose to beta-anomers.
본 발명에서 용어, "GAL7"은 갈락토스 이용에 관여하는 유전자 중 하나로, 갈락토스-1-인산 우리딜 전이 효소(Galactose-1-phosphate uridyl transferase)를 코딩하는 유전자를 의미한다. 상기 갈락토스-1-인산 우리딜 전이 효소는 갈락토스 대사의 두 번째 단계에서 UDP-D-글루코스 및 알파-D-갈락토스-1-인산으로부터 글루코스-1-인산 및 UDP-갈락토스를 합성하는 효소이다.As used herein, the term "GAL7" refers to a gene encoding galactose-1-phosphate uridyl transferase, which is one of the genes involved in the utilization of galactose. The galactose-1-phosphate uridyl transferase is an enzyme that synthesizes glucose-1-phosphate and UDP-galactose from UDP-D-glucose and alpha-D-galactose-1-phosphate in the second step of galactose metabolism.
본 발명에서 용어, "GAL2"는 갈락토스 이용에 관여하는 유전자 중 하나로, 갈락토스 퍼미아제(Galactose permease)로도 명명되는 갈락토스 및 글루코오스 막 횡단 수송체(Galactose and glucose transmembrane transporter)를 코딩하는 유전자를 의미한다. 상기 갈락토스 퍼미아제는 탄소원인 갈락토스를 세포 내로 유입시켜 대사 될 수 있도록 하며, 또한 포도당을 수송할 수 있다.As used herein, the term "GAL2" is one of the genes involved in galactose utilization, and refers to a gene encoding a galactose and glucose transmembrane transporter, also referred to as galactose permease. . The galactose permease allows galactose, a carbon source, to be introduced into cells to be metabolized, and can also transport glucose.
본 발명에 있어서, 상기 GAL80, GAL1, GAL10, GAL7 또는 GAL2 유전자는 미생물 유래일 수 있고, 구체적으로 효모 유래일 수 있으며, 보다 구체적으로는 사카로마이세스 속(Saccharomyces sp.) 및 클루이베로마이세스(Kluyveromyce sp.)속 유래일 수 있고, 보다 더욱 구체적으로는 사카로마이세스 세레비지에(Saccharomyces cerevisiae) 유래일 수 있으나, 이에 제한되지 않는다.In the present invention, the GAL80, GAL1, GAL10, GAL7 or GAL2 genes may be derived from microorganisms, specifically from yeast, and more specifically from the genus Saccharomyces ( Saccharomyces sp.) and Kluyveromyces ( Kluyveromyce sp.) It may be derived from, and more specifically, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) It may be derived, but is not limited thereto.
상기 GAL80, GAL1, GAL10, GAL7 또는 GAL2 유전자는 서열번호 1 또는 서열번호 2 또는 서열번호 3 또는 서열번호 4 또는 서열번호 5로 기재된 염기서열을 가지거나, 포함하거나, 이루어지거나, 상기 염기서열로 필수적으로 이루어질(essentially consisting of) 수 있다.The GAL80, GAL1, GAL10, GAL7 or GAL2 gene has, contains, consists of, or is essential to the nucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 It may consist essentially of.
또한, 상기 GAL80, GAL1, GAL10, GAL7 또는 GAL2 유전자는 서열번호 1 또는 서열번호 2 또는 서열번호 3 또는 서열번호 4 또는 서열번호 5로 기재된 염기서열과 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% 또는 99.9% 이상의 상동성 또는 동일성을 가지는 염기서열을 포함할 수 있다. 또한, 이러한 상동성 또는 동일성을 가지며 본 발명의 상기 GAL80, GAL1, GAL10, GAL7 또는 GAL2 유전자에 상응하는 효능을 나타내는 염기서열이라면, 일부 서열이 결실, 변형, 치환, 보존적 치환 또는 부가된 염기서열을 갖는 폴리뉴클레오티드도 본 발명의 범위 내에 포함됨은 자명하다. In addition, the GAL80, GAL1, GAL10, GAL7 or GAL2 gene is at least 80%, 85%, 90%, 95% of the nucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 , 96%, 97%, 98%, 99%, 99.5%, 99.7%, or may include a base sequence having a homology or identity of 99.9% or more. In addition, if a nucleotide sequence having such homology or identity and exhibiting an efficacy corresponding to the GAL80, GAL1, GAL10, GAL7 or GAL2 gene of the present invention, some sequences may be deleted, modified, substituted, conservatively substituted, or added nucleotide sequences It is obvious that polynucleotides having are also included within the scope of the present invention.
예를 들어, 상기 염기서열 말단 그리고/또는 내부에 본 발명의 상기 GAL80, GAL1, GAL10, GAL7 또는 GAL2 유전자의 기능을 변경하지 않는 서열 추가 또는 결실, 자연적으로 발생할 수 있는 돌연변이, 잠재성 돌연변이(silent mutation) 또는 상기 유전자로부터 코딩된 아미노산 서열에서 보존적 치환을 가지는 경우이다.For example, sequence addition or deletion, naturally occurring mutation, latent mutation (silent mutation) or a conservative substitution in the amino acid sequence encoded from the gene.
상기 “보존적 치환(conservative substitution)”은 한 아미노산을 유사한 구조적 및/또는 화학적 성질을 갖는 또 다른 아미노산으로 치환시키는 것을 의미한다. 이러한 아미노산 치환은 일반적으로 잔기의 극성, 전하, 용해도, 소수성, 친수성 및/또는 양친매성(amphipathic nature)에서의 유사성에 근거하여 발생할 수 있다. 통상적으로, 보존적 치환은 단백질 또는 폴리펩티드의 활성에 거의 영향을 미치지 않거나 또는 영향을 미치지 않을 수 있다.The "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions can generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.
본 발명에서 용어, ‘상동성(homology)’ 또는 ‘동일성(identity)’은 두 개의 주어진 아미노산 서열 또는 염기 서열 상호간 유사한 정도를 의미하며 백분율로 표시될 수 있다. 용어 상동성 및 동일성은 종종 상호교환적으로 이용될 수 있다.In the present invention, the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or base sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably.
보존된(conserved) 폴리뉴클레오티드 또는 폴리펩티드의 서열 상동성 또는 동일성은 표준 배열 알고리즘에 의해 결정되며, 사용되는 프로그램에 의해 확립된 디폴트 갭 페널티가 함께 이용될 수 있다. 실질적으로, 상동성을 갖거나(homologous) 또는 동일한(identical) 서열은 일반적으로 서열 전체 또는 일부분과 중간 또는 높은 엄격한 조건(stringent conditions)에서 하이브리드할 수 있다. 하이브리드화는 폴리뉴클레오티드에서 일반 코돈 또는 코돈 축퇴성을 고려한 코돈을 함유하는 폴리뉴클레오티드와의 하이브리드화 역시 포함됨이 자명하다.Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms, together with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of the sequence under moderate or high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing common codons or codons in consideration of codon degeneracy in polynucleotides.
임의의 두 폴리뉴클레오티드 또는 폴리펩티드 서열이 상동성, 유사성 또는 동일성을 갖는지 여부는, 예를 들어, Pearson et al(1988) [Proc. Natl. Acad. Sci. USA 85]: 2444에서와 같은 디폴트 파라미터를 이용하여 "FASTA" 프로그램과 같은 공지의 컴퓨터 알고리즘을 이용하여 결정될 수 있다. 또는, EMBOSS 패키지의 니들만 프로그램(EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277)(버전 5.0.0 또는 이후 버전)에서 수행되는 바와 같은, 니들만-운치(Needleman-Wunsch) 알고리즘(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453)이 사용되어 결정될 수 있다(GCG 프로그램 패키지 (Devereux, J., et al, Nucleic Acids Research 12: 387(1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994, 및 [CARILLO ETA/.](1988) SIAM J Applied Math 48: 1073을 포함한다). 예를 들어, 국립 생물공학 정보 데이터베이스 센터의 BLAST, 또는 ClustalW를 이용하여 상동성, 유사성 또는 동일성을 결정할 수 있다.Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: can be determined using known computer algorithms such as the “FASTA” program using default parameters as in 2444. or, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), It can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073. For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity or identity.
폴리뉴클레오티드 또는 폴리펩티드의 상동성, 유사성 또는 동일성은, 예를 들어, Smith and Waterman, Adv. Appl. Math(1981) 2:482 에 공지된 대로, 예를 들면, Needleman et al.(1970), J Mol Biol. 48:443과 같은 GAP 컴퓨터 프로그램을 이용하여 서열 정보를 비교함으로써 결정될 수 있다. 요약하면, GAP 프로그램은 두 서열 중 더 짧은 것에서의 기호의 전체 수로, 유사한 배열된 기호(즉, 뉴클레오티드 또는 아미노산)의 수를 나눈 값으로 정의할 수 있다. GAP 프로그램을 위한 디폴트 파라미터는 (1) 이진법 비교 매트릭스(동일성을 위해 1 그리고 비-동일성을 위해 0의 값을 함유함) 및 Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358(1979)에 의해 개시된 대로, Gribskov et al(1986) Nucl. Acids Res. 14: 6745의 가중된 비교 매트릭스 (또는 EDNAFULL (NCBI NUC4.4의 EMBOSS 버전) 치환 매트릭스); (2) 각 갭을 위한 3.0의 페널티 및 각 갭에서 각 기호를 위한 추가의 0.10 페널티 (또는 갭 개방 패널티 10, 갭 연장 패널티 0.5); 및 (3) 말단 갭을 위한 무 페널티를 포함할 수 있다.Homology, similarity or identity of polynucleotides or polypeptides can be found in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. It can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, the GAP program can define the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program are (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or 10 gap opening penalty, 0.5 gap extension penalty); and (3) no penalty for end gaps.
본 발명에 있어서, 효모는 갈락토스 발효능(galactose-fermenting) 또는 락토스 발효능(lactose-fermenting)을 갖는 것일 수 있다.In the present invention, yeast may have galactose-fermenting ability or lactose-fermenting ability.
구체적으로, 상기 효모는 갈락토스 발효능(galactose-fermenting) 또는 락토스 발효능(lactose-fermenting)을 갖는 사카로마이세스 속(Saccharomyces sp.) 및 클루이베로마이세스(Kluyveromyce sp.)속일 수 있고, 보다 구체적으로는 갈락토스 발효능(galactose-fermenting) 또는 락토스 발효능(lactose-fermenting)을 갖는 사카로마이세스 세레비지에(Saccharomyces cerevisiae)일 수 있다.Specifically, the yeast may belong to the genus Saccharomyces sp. and Kluyveromyce sp. having galactose-fermenting or lactose-fermenting ability, and more Specifically, it may be Saccharomyces cerevisiae having galactose-fermenting or lactose-fermenting ability.
본 발명에 있어서, 효모 변이주는 전술한 효모에서 GAL80, GAL1, GAL10, GAL7 및 GAL2 유전자가 결손된 효모를 의미하는 것일 수 있다.In the present invention, yeast mutants may mean yeast in which GAL80, GAL1, GAL10, GAL7, and GAL2 genes are deleted from the yeast described above.
상기 GAL80, GAL1, GAL10, GAL7 및 GAL2 유전자를 결손시키는 방법은 CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)-CAS9 방법일 수 있으나, 당업계에 알려진 유전자 결손 방법이라면 제한 없이 사용할 수 있다.The method for deleting the GAL80, GAL1, GAL10, GAL7 and GAL2 genes may be a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) -CAS9 method, but any gene deletion method known in the art may be used without limitation.
상기 결손 부위는 결손 시 GAL80, GAL1, GAL10, GAL7 및 GAL2 유전자의 발현이 이루어지지 않는 GAL80, GAL1, GAL10, GAL7 및 GAL2 유전자 내 서열이라면 특별히 제한되지 않으나, 구체적으로 GAL80 유전자(서열번호 1)의 프로모터 및 코딩서열(Coding sequence)의 일부, GAL1 유전자(서열번호 2)의 프로모터 및 코딩서열 일부, GAL10 유전자(서열번호 3)의 프로모터 및 코딩서열 일부, GAL7 유전자(서열번호 4)의 프로모터 및 코딩서열 일부와, GAL2 유전자(서열번호 5)의 프로모터 및 코딩서열 일부가 결손된 것일 수 있다.The deletion site is not particularly limited as long as it is a sequence within the GAL80, GAL1, GAL10, GAL7, and GAL2 genes that do not express the GAL80, GAL1, GAL10, GAL7, and GAL2 genes when the deletion occurs, but is specifically a sequence of the GAL80 gene (SEQ ID NO: 1). Part of promoter and coding sequence, part of promoter and coding sequence of GAL1 gene (SEQ ID NO: 2), part of promoter and coding sequence of GAL10 gene (SEQ ID NO: 3), promoter and coding sequence of GAL7 gene (SEQ ID NO: 4) Part of the sequence, and part of the promoter and coding sequence of the GAL2 gene (SEQ ID NO: 5) may be missing.
본 발명의 일 구현예에서, 상기 GAL80, GAL1, GAL10, GAL7 및 GAL2 유전자의 결손은 서열번호 6의 서열을 제거함으로써 GAL1, GAL10, GAL7 유전자들은 한꺼번에 결실시킨 후, 서열번호 7의 서열을 제거하여 GAL2 유전자를 결실시킴으로써 이루어졌다.In one embodiment of the present invention, the GAL80, GAL1, GAL10, GAL7, and GAL2 genes are deleted by deleting the sequence of SEQ ID NO: 6, thereby deleting the GAL1, GAL10, and GAL7 genes at once, and then removing the sequence of SEQ ID NO: 7. This was done by deleting the GAL2 gene.
본 발명에 있어서, 효모 변이주는 갈락토스 결핍 배지에서 균주 성장이 우수한 것일 수 있다.In the present invention, the yeast mutant strain may have excellent strain growth in a galactose deficient medium.
본 발명의 일 구현예에서, 재조합 단백질 발현 벡터인 YGa-T3CalB14 벡터로 형질전환 된 gal80 균주(GAL80 유전자 단독 결손 균주)와 YGa-T3CalB14 벡터로 형질전환 된 본 발명의 효모 변이주(도 6에서 gal로 표시)를 갈락토스 결핍 배지에서 배양시, 갈락토스 1% 함유 배지에서 배양한 YGa-T3CalB14 벡터로 형질전환된 야생형 균주보다 성장이 약 10-15% 더 우수하였다(도 6). In one embodiment of the present invention, the gal80 strain transformed with the YGa-T3CalB14 vector, which is a recombinant protein expression vector (GAL80 gene alone defective strain), and the yeast mutant strain of the present invention transformed with the YGa-T3CalB14 vector (gal in FIG. 6) When cultured in a galactose-deficient medium, the growth was about 10-15% better than that of the wild-type strain transformed with the YGa-T3CalB14 vector cultured in a medium containing 1% galactose (FIG. 6).
본 발명에 있어서, 효모 변이주는 재조합 단백질 발현 벡터로 형질전환되어 재조합 단백질을 고수율로 발현하는 것일 수 있다.In the present invention, the yeast mutant may be transformed with a recombinant protein expression vector to express the recombinant protein in high yield.
상기 벡터는 상기 폴리뉴클레오티드를 숙주 세포에서 발현시키기 위한 발현 벡터일 수 있으나, 이에 제한되지 않는다. 본 발명의 목적상, 상기 숙주 세포는 진핵세포일 수 있고, 구체적으로 효모일 수 있으며, 이에 대해서는 전술한 바와 같다.The vector may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto. For the purposes of the present invention, the host cell may be a eukaryotic cell, specifically yeast, as described above.
본 발명의 벡터는 적합한 숙주 내에서 목적 폴리펩티드를 발현시킬 수 있도록 적합한 발현조절영역(또는 발현조절서열)에 작동 가능하게 연결된 상기 목적 폴리펩티드를 코딩하는 폴리뉴클레오티드의 염기서열을 포함하는 DNA 제조물을 포함할 수 있다. 상기 발현조절영역은 전사를 개시할 수 있는 프로모터, 그러한 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합부위를 코딩하는 서열, 및 전사 및 해독의 종결을 조절하는 서열을 포함할 수 있다. 벡터는 적당한 숙주 세포 내로 형질전환된 후, 숙주 게놈과 무관하게 복제되거나 기능할 수 있으며, 게놈 그 자체에 통합될 수 있다.The vector of the present invention may include a DNA product containing the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to express the target polypeptide in a suitable host. can The expression control region may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can integrate into the genome itself.
본 발명에서 사용되는 벡터는 특별히 한정되지 않으며, 당업계에 알려진 임의의 벡터를 이용할 수 있다. 통상 사용되는 벡터의 예로는 천연 상태이거나 재조합된 상태의 플라스미드, 코스미드, 바이러스 및 박테리오파지를 들 수 있다. Vectors used in the present invention are not particularly limited, and any vectors known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages.
본 발명의 숙주 세포가 진핵세포 일 경우, 예를 들어, 파지 벡터 또는 코스미드 벡터로서 pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, 및 Charon21A 등을 사용할 수 있으며, 플라스미드 벡터로서 pRS416, pRS426, pRS415, pRS425, YEp13, YEp24, YCp50, pBR계, pUC계, pBluescriptII계, pGEM계, pTZ계, pCL계 및 pET계 등을 사용할 수 있으나, 이에 한정되지 않는다.When the host cell of the present invention is a eukaryotic cell, for example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, As plasmid vectors, pRS416, pRS426, pRS415, pRS425, YEp13, YEp24, YCp50, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. may be used, but are not limited thereto.
본 발명의 숙주 세포가 효모세포 일 경우, 효모 발현 벡터는 염색체 삽입 플라스미드(YIp: integrative yeast plasmid) 및 염색체 외 플라스미드 벡터(extrachromosomal plasmid vector)가 모두 가능하다. 상기 염색체 외 플라스미드 벡터는 에피솜 효모 플라스미드(YEp: episomal yeast plasmid), 복제 효모 플라스미드(YRp: replicative yeast plasmid) 및 효모 중심체 플라스미드(YCp: yeast centromere plasmid)를 포함할 수 있다. 또한, 인위적 효모 염색체들(YACs: artificial yeast chromosomes)도 본 발명의 벡터로 이용이 가능하다. 구체적인 예로서, 이용 가능한 벡터는 pYG, pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPα A, pGAPα B, pGAPα C, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6α A, pPIC6α B, pPIC6α C, pPIC9K, pYC2/CT, pYD1 Yeast Display Vector, pYES2, pYES2/CT, pYES2/NT A, pYES2/NT B, pYES2/NT C, pYES2/CT, pYES2.1, pYES-DEST52, pTEF1/Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A form, pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405 및 pYJ406를 포함할 수 있으나, 이에 제한되는 것은 아니다.When the host cell of the present invention is a yeast cell, the yeast expression vector may be either an integrative yeast plasmid (YIp) or an extrachromosomal plasmid vector. The extrachromosomal plasmid vector may include an episomal yeast plasmid (YEp), a replicative yeast plasmid (YRp), and a yeast centromere plasmid (YCp). In addition, artificial yeast chromosomes (YACs) can also be used as vectors of the present invention. As specific examples, available vectors are pYG, pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPα A, pGAPα B, pGAPα C, pPIC3 .5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6α A, pPIC6α B, pPIC6α C, pPIC9K, pYC2/CT, pYD1 Yeast Display Vector, pYES2, pYES2/CT, pYES2/NT A, pYES2/NT B, pYES2/ NT C, pYES2/CT, pYES2.1, pYES-DEST52, pTEF1/Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP47 , pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A form, pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405 and pYJ406, but is not limited thereto.
일례로 세포 내 염색체 삽입용 벡터를 통해 목적 폴리펩티드를 코딩하는 폴리뉴클레오티드를 염색체 내로 삽입할 수 있다. 상기 폴리뉴클레오티드의 염색체 내로의 삽입은 당업계에 알려진 임의의 방법, 예를 들면, 상동 유전자 재조합(homologous recombination)에 의하여 이루어질 수 있으나, 이에 한정되지는 않는다. 상기 염색체 삽입 여부를 확인하기 위한 선별 마커(selection marker)를 추가로 포함할 수 있다. 상기 선별 마커는 벡터로 형질전환 된 세포를 선별, 즉 목적 핵산 분자의 삽입 여부를 확인하기 위한 것으로, 약물 내성, 영양 요구성, 세포 독성제에 대한 내성 또는 표면 폴리펩티드의 발현과 같은 선택가능 표현형을 부여하는 마커들이 사용될 수 있다. 선택제(selective agent)가 처리된 환경에서는 선별 마커를 발현하는 세포만 생존하거나 다른 표현 형질을 나타내므로, 형질전환 된 세포를 선별할 수 있다.For example, a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for chromosomal insertion into a cell. Insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for determining whether the chromosome is inserted may be further included. The selectable marker is used to select cells transformed with a vector, that is, to determine whether a target nucleic acid molecule has been inserted, and can exhibit selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. markers may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive or exhibit other phenotypes, and thus transformed cells can be selected.
본 발명에서 용어 "형질전환"은 표적 폴리펩티드를 코딩하는 폴리뉴클레오티드를 포함하는 벡터를 숙주 세포 혹은 미생물 내에 도입하여 숙주 세포 내에서 상기 폴리뉴클레오티드가 코딩하는 폴리펩티드가 발현할 수 있도록 하는 것을 의미한다. 형질전환 된 폴리뉴클레오티드는 숙주 세포 내에서 발현될 수 있기만 한다면, 숙주 세포의 염색체 내에 삽입되어 위치하거나 염색체 외에 위치하거나 상관없이 이들 모두를 포함할 수 있다. 또한, 상기 폴리뉴클레오티드는 목적 폴리펩티드를 코딩하는 DNA 및/또는 RNA를 포함한다. 상기 폴리뉴클레오티드는 숙주 세포 내로 도입되어 발현될 수 있는 것이면, 어떠한 형태로도 도입될 수 있다. 예를 들면, 상기 폴리뉴클레오티드는 자체적으로 발현되는데 필요한 모든 요소를 포함하는 유전자 구조체인 발현 카세트(expression cassette)의 형태로 숙주 세포에 도입될 수 있다. 상기 발현 카세트는 통상 상기 폴리뉴클레오티드에 작동 가능하게 연결되어 있는 프로모터(promoter), 전사 종결 신호, 리보좀 결합 부위 및 번역 종결 신호를 포함할 수 있다. 상기 발현 카세트는 자체 복제가 가능한 발현 벡터 형태일 수 있다. 또한, 상기 폴리뉴클레오티드는 그 자체의 형태로 숙주 세포에 도입되어 숙주 세포에서 발현에 필요한 서열과 작동 가능하게 연결되어 있는 것일 수도 있으며, 이에 제한되지 않는다.In the present invention, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it may be inserted into and located in the chromosome of the host cell or located outside the chromosome, both of which may be included. In addition, the polynucleotide includes DNA and/or RNA encoding a polypeptide of interest. The polynucleotide may be introduced in any form as long as it can be introduced into and expressed in a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a genetic construct containing all elements required for self-expression. The expression cassette may typically include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.
본 발명의 벡터를 형질전환 시키는 방법은 핵산을 세포 내로 도입하는 어떤 방법도 포함되며, 숙주 세포에 따라 당 분야에서 공지된 바와 같이 적합한 표준 기술을 선택하여 수행할 수 있다. 예를 들어, 전기천공법(electroporation), 인산칼슘(CaPO4) 침전, 염화칼슘(CaCl2) 침전, 미세주입법(microinjection), 폴리에틸렌 글리콜(PEG)법, DEAE-덱스트란법, 양이온 리포좀법, 및 초산 리튬-DMSO법 등이 있으나, 이에 제한되지 않는다.The method of transforming the vector of the present invention includes any method of introducing a nucleic acid into a cell, and can be performed by selecting an appropriate standard technique as known in the art according to the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposomal method, and lithium acetate -DMSO method, etc., but is not limited thereto.
또한, 상기에서 용어 "작동 가능하게 연결"된 것이란 목적 폴리펩티드를 코딩하는 폴리뉴클레오티드의 전사를 개시 및 매개하도록 하는 프로모터 서열과 상기 폴리뉴클레오티드 서열이 기능적으로 연결되어 있는 것을 의미한다.In addition, the term "operably linked" as used herein means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target polypeptide.
본 발명의 목적상, 상기 재조합 단백질 발현 벡터는 GAL1, GAL10, GAL7 및 GAL2로 이루어지는 군으로부터 선택되는 어느 하나 이상의 유전자의 프로모터를 포함할 수 있고, 구체적으로 GAL10 유전자의 프로모터를 포함할 수 있으나, 이에 제한되지 않는다.For the purpose of the present invention, the recombinant protein expression vector may include a promoter of one or more genes selected from the group consisting of GAL1, GAL10, GAL7, and GAL2, and may specifically include a promoter of the GAL10 gene. Not limited.
본 발명의 일 구현예에서, GAL10 프로모터를 포함하는 재조합 단백질 발현 벡터인 YGa-T3CalB14 벡터로 형질전환 된 야생형 균주를 기준으로 보았을 때, YGa-T3CalB14 벡터로 형질전환 된 gal1 변이주(GAL1 유전자 단독 결손 균주), YGa-T3CalB14 벡터로 형질전환된 gal1,10,7 변이주(GAL1, GAL10 및 GAL7 유전자 결손 균주) 및 TDH3(glyceraldehyde 3-phosphate dehydrogenase) 프로모터를 포함하는 재조합 단백질 발현 벡터인 YG-T3CalB14 벡터로 형질전환 된 야생형 균주는 성장이 느리고, YGa-T3CalB14 벡터로 형질전환 된 gal80 균주(GAL80 유전자 단독 결손 균주)와 YGa-T3CalB14 벡터로 형질전환 된 본 발명의 효모 변이주(도 6에서 gal로 표시)는 성장이 약 10-15% 더 우수함을 확인하였다(도 6).In one embodiment of the present invention, based on the wild-type strain transformed with the YGa-T3CalB14 vector, which is a recombinant protein expression vector containing the GAL10 promoter, the gal1 mutant strain transformed with the YGa-T3CalB14 vector (GAL1 gene alone deletion strain ), gal1,10,7 mutants (GAL1, GAL10 and GAL7 gene deletion strains) transformed with YGa-T3CalB14 vector and YG-T3CalB14 vector, a recombinant protein expression vector containing TDH3 (glyceraldehyde 3-phosphate dehydrogenase) promoter The transformed wild-type strain grew slowly, and the gal80 strain transformed with the YGa-T3CalB14 vector (strain lacking the GAL80 gene alone) and the yeast mutant strain of the present invention transformed with the YGa-T3CalB14 vector (indicated by gal in FIG. 6) showed growth It was confirmed that this was about 10-15% better (FIG. 6).
이로부터 GAL1 유전자 단독 결손 균주, GAL1, GAL10 및 GAL7 유전자 결손 균주와 본 발명의 효모 변이주를 동일한 재조합 단백질 발현 벡터로 각각 형질전환 시 본 발명의 효모 변이주의 균주 성장이 가장 우수함을 알 수 있다. 뿐만 아니라, GAL10 프로모터를 포함하는 재조합 단백질 발현 벡터로 형질전환 된 야생형 균주가 구성적 프로모터인 TDH3 프로모터를 포함하는 재조합 단백질 발현 벡터로 형질전환 된 야생형 균주보다 균주 성장이 우수함을 알 수 있다.From this, it can be seen that the strain growth of the yeast mutant strain of the present invention is the best when transforming the GAL1 gene alone defective strain, GAL1, GAL10 and GAL7 gene defective strains and the yeast mutant strain of the present invention with the same recombinant protein expression vector. In addition, it can be seen that the growth of the wild-type strain transformed with the recombinant protein expression vector containing the GAL10 promoter is superior to the wild-type strain transformed with the recombinant protein expression vector containing the constitutive promoter TDH3 promoter.
또한, 시간별로 분비된 CalB14의 양을 SDS-PAGE로 비교한 결과, 균주 성장과 비례하는 발현량을 나타내었으며(도 7), 각 균주로부터 발현되어 배양액에 포함된 리파제의 활성을 확인한 결과, GAL80 유전자 단독 결손 균주와 본 발명의 효모 변이주에서 발현된 리파제의 활성이 가장 높음을 확인하였는 바(도 8), 이로부터 사카로마이세스 세레비지에 균주에서 재조합 단백질을 발현하는 경우 GAL10 프로모터를 포함하는 재조합 단백질 발현 벡터를 GAL80 유전자 단독 결손 균주 또는 본 발명의 효모 변이주에 형질전환하여 재조합 단백질을 발현시키는 것이 균주 성장과 재조합 단백질 발현에 있어 최적 조건임을 알 수 있다.In addition, as a result of comparing the amount of CalB14 secreted over time by SDS-PAGE, the expression level was proportional to the growth of the strain (FIG. 7), and as a result of confirming the activity of lipase contained in the culture medium expressed from each strain, GAL80 It was confirmed that the activity of lipase expressed in the gene-only defective strain and the yeast mutant strain of the present invention was the highest (Fig. 8). From this, when the recombinant protein was expressed in the Saccharomyces cerevisiae strain, It can be seen that transforming the recombinant protein expression vector into the GAL80 gene-only defective strain or the yeast mutant strain of the present invention to express the recombinant protein is an optimal condition for strain growth and recombinant protein expression.
다만, 상기 구현예에서는 GAL80 유전자 단독 결손 균주와 본 발명의 효모 변이주 간의 균주 성장 및 재조합 단백질 발현량 차이를 확인할 수 없었는 바, 유가식 배양을 통해 그 차이를 확인한 결과, GAL80 유전자 단독 결손 균주는 48시간 동안 발효 후 OD600 143까지 성장한 반면 본 발명의 효모 변이주는 이보다 21% 증가된 172까지 성장하였으며(도 9A), 분비 생산된 단백질의 양은 본 발명의 효모 변이주가 0.63g/L로 GAL80 유전자 단독 결손 균주(0.56g/L)에 비해 12% 증가하여(도 9B), 본 발명의 효모 변이주가 GAL80 유전자 단독 결손 균주 대비 우수한 발현량을 나타냄을 확인하였다. However, in the above embodiment, it was not possible to confirm the difference in strain growth and recombinant protein expression between the GAL80 gene-only defective strain and the yeast mutant strain of the present invention. As a result of confirming the difference through fed-batch culture, the GAL80 gene-only defective strain was 48 After fermentation for a period of time, the OD 600 grew to 143, whereas the yeast mutant strain of the present invention grew to 172, a 21% increase (Fig. 9A), and the amount of secreted protein produced was 0.63 g/L for the yeast mutant strain of the present invention, which was the GAL80 gene alone. It increased by 12% compared to the defective strain (0.56 g / L) (FIG. 9B), confirming that the yeast mutant strain of the present invention exhibited an excellent expression level compared to the GAL80 gene-only defective strain.
또한, 목적 단백질의 종류를 달리하여 성장 및 재조합 단백질 발현량을 확인하였을 시에도 본 발명의 효모 변이주는 목적 단백질의 종류와 무관하게 GAL80 유전자 단독 결손 균주보다 성장이 21-38% 개선되고 분비 생산되는 재조합 단백질의 양도 12-22% 증가함을 확인하였다(도 10).In addition, even when the growth and recombinant protein expression levels were confirmed by varying the type of target protein, the yeast mutant strain of the present invention showed 21-38% improvement in growth and secretion production compared to strains lacking the GAL80 gene alone, regardless of the type of target protein. It was confirmed that the amount of recombinant protein increased by 12-22% (FIG. 10).
전술한 바와 같이, 본 발명은 산업적으로 적용 가능한 재조합 단백질 생산 균주를 개발하기 위해 사카로마이세스 세레비지에의 갈락토스 이용에 관여하는 모든 유전자가 결손된 변이주를 제조하였으며, 유가식 배양시 야생형 균주 또는 종래 GAL80 유전자 단독 결손 균주 대비 현저한 균주 성장 및 재조합 단백질 생산 효과를 최초로 확인한 것에 의의가 있다.As described above, the present invention prepared a mutant strain in which all genes involved in the use of galactose in Saccharomyces cerevisiae were deleted in order to develop an industrially applicable recombinant protein production strain, and wild-type strain or It is significant that it was the first time that significant strain growth and recombinant protein production effects were confirmed compared to the conventional strains lacking the GAL80 gene alone.
본 발명의 다른 하나의 양태는 재조합 단백질 생산 방법을 제공한다.Another aspect of the present invention provides a method for producing a recombinant protein.
구체적으로, 상기 방법은 a) 상기 효모 변이주에, 외래 목적 단백질을 코딩하는 유전자를 포함하는 재조합 단백질 발현 벡터를 형질전환하는 단계; 및 b) 상기 a)의 형질전환된 효모 변이주를 갈락토스 결핍 배지에서 배양하고 재조합 단백질을 발현시키는 단계;를 포함하는 것일 수 있다.Specifically, the method includes a) transforming the yeast mutant strain with a recombinant protein expression vector containing a gene encoding a foreign protein of interest; and b) culturing the transformed yeast mutant of a) in a galactose-deficient medium and expressing the recombinant protein.
여기에서 사용된 용어는 전술한 바와 같다.Terms used herein are as described above.
상기 a) 단계는 상기 효모 변이주에 외래 목적 단백질을 코딩하는 유전자를 포함하는 재조합 단백질 발현 벡터를 형질전환하는 것일 수 있다.Step a) may be to transform the yeast mutant strain with a recombinant protein expression vector containing a gene encoding a foreign protein of interest.
상기 벡터는 GAL1, GAL10, GAL7 및 GAL2로 이루어지는 군으로부터 선택되는 어느 하나 이상의 유전자의 프로모터를 포함할 수 있고, 구체적으로 GAL10 유전자의 프로모터를 포함할 수 있으나, 이에 제한되지 않으며, 이에 대해서는 전술한 바와 같다.The vector may include a promoter of one or more genes selected from the group consisting of GAL1, GAL10, GAL7, and GAL2, and may specifically include a promoter of the GAL10 gene, but is not limited thereto, as described above. same.
상기 형질전환 방법은 당업계에서 사용되는 방법이라면 제한 없이 사용할 수 있으며, 이에 대해서는 전술한 바와 같다.The transformation method can be used without limitation as long as it is a method used in the art, as described above.
상기 목적 단백질은 효모에서 발현 가능한 모든 단백질일 수 있다. 일예로, 상기 목적 단백질은 리파제(lipase), 자일라나제(Xylanase), 피타아제(Phytase), 인슐린, 단일 체인(chain) 항체 및 EGF(Epidermal Growth Factor) 등일 수 있으나, 이에 제한되지 않고 의학적, 산업적으로 유용한 재조합 단백질을 모두 포함할 수 있다.The target protein may be any protein that can be expressed in yeast. For example, the target protein may be lipase, xylanase, phytase, insulin, single chain antibody, and EGF (Epidermal Growth Factor), but is not limited thereto. It may include all industrially useful recombinant proteins.
상기 b) 단계는 상기 a) 단계의 형질전환 된 효모 변이주를 갈락토스 결핍 배지에서 배양하고 재조합 단백질을 발현시키는 것일 수 있다.Step b) may include culturing the yeast mutant transformed in step a) in a galactose-deficient medium and expressing the recombinant protein.
상기 배양에 사용되는 배지 및 기타 배양 조건은 통상의 사카로마이세스 속 미생물의 배양에 사용되는 배지라면 특별한 제한 없이 어느 것이나 사용될 수 있으며, 구체적으로는 본 발명의 균주를 적당한 탄소원, 질소원, 인원, 무기 화합물, 아미노산 및/또는 비타민 등을 함유한 통상의 배지 내에서 호기성 또는 혐기성 조건 하에서 온도, pH 등을 조절하면서 배양할 수 있다.Any medium and other culture conditions used for the culture may be used without particular limitation as long as they are medium used for the culture of ordinary microorganisms of the genus Saccharomyces. It can be cultured while controlling temperature, pH, etc. under aerobic or anaerobic conditions in a conventional medium containing inorganic compounds, amino acids, and/or vitamins.
본 발명에서 상기 탄소원으로는 글루코오스, 프룩토오스, 수크로오스, 말토오스 등과 같은 탄수화물; 만니톨, 소르비톨 등과 같은 당 알코올, 피루브산, 락트산, 시트르산 등과 같은 유기산; 글루탐산, 메티오닌, 라이신 등과 같은 아미노산 등이 포함될 수 있으나, 이에 제한되지 않는다. 또한, 전분 가수분해물, 당밀, 블랙스트랩 당밀, 쌀겨울, 카사버, 사탕수수 찌꺼기 및 옥수수 침지액 같은 천연의 유기 영양원을 사용할 수 있으며, 글루코오스 및 살균된 전처리 당밀(즉, 환원당으로 전환된 당밀) 등과 같은 탄수화물이 사용될 수 있고, 그 외의 적정량의 탄소원을 제한 없이 다양하게 이용할 수 있다. 이들 탄소원은 단독으로 사용되거나 2 종 이상이 조합되어 사용될 수 있다.Examples of the carbon source in the present invention include carbohydrates such as glucose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included, but are not limited thereto. In addition, natural organic nutritional sources such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sorghum and corn steep liquor can be used, as well as glucose and pasteurized pretreated molasses (i.e., molasses converted to reducing sugars). Carbohydrates such as may be used, and various other carbon sources in appropriate amounts may be used without limitation. These carbon sources may be used alone or in combination of two or more.
상기 질소원으로는 암모니아, 황산암모늄, 염화암모늄, 초산암모늄, 인산암모늄, 탄산안모늄, 질산암모늄 등과 같은 무기질소원; 아미노산, 펩톤, NZ-아민, 육류 추출물, 효모 추출물, 맥아 추출물, 옥수수 침지액, 카세인 가수분해물, 어류 또는 그의 분해생성물, 탈지 대두 케이크 또는 그의 분해 생성물 등과 같은 유기 질소원이 사용될 수 있다. 이들 질소원은 단독으로 사용되거나 2 종 이상이 조합되어 사용될 수 있으나, 이에 제한되지 않는다.Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids, peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysates, fish or degradation products thereof, defatted soybean cakes or degradation products thereof, and the like can be used. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.
상기 인원으로는 인산 제1칼륨, 인산 제2칼륨, 또는 이에 대응되는 소디움-함유 염 등이 포함될 수 있다. 무기화합물로는 염화나트륨, 염화칼슘, 염화철, 황산마그네슘, 황산철, 황산망간, 탄산칼슘 등이 사용될 수 있다.The number of persons may include monopotassium phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used.
그 외에 상기 배지에는 아미노산, 비타민 및/또는 적절한 전구체 등이 포함될 수 있다.In addition, amino acids, vitamins, and/or appropriate precursors may be included in the medium.
상기 배지 또는 전구체는 배양물에 회분식 또는 연속식으로 첨가될 수 있으며, 이에 제한되지 않는다.The medium or precursor may be added to the culture in a batch or continuous manner, but is not limited thereto.
본 발명에서, 균주의 배양 중에 수산화암모늄, 수산화칼륨, 암모니아, 인산, 황산 등과 같은 화합물을 배양물에 적절한 방식으로 첨가하여, 배양물의 pH를 조정할 수 있다. 또한, 배양 중에는 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 또한, 배양물의 호기 상태를 유지하기 위하여, 배양물 내로 산소 또는 산소 함유 기체를 주입하거나 혐기 및 미호기 상태를 유지하기 위해 기체의 주입 없이 혹은 질소, 수소 또는 이산화탄소 가스를 주입할 수 있다.In the present invention, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid and the like to the culture in an appropriate manner during cultivation of the strain. In addition, during cultivation, the formation of bubbles can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen or carbon dioxide gas may be injected without injection of gas or gas to maintain the anaerobic and non-aerobic state.
배양물의 온도는 25℃내지 40℃일 수 있으며, 보다 구체적으로는 28℃내지 37℃일 수 있으나 이에 제한되지 않는다. 배양 기간은 원하는 유용 물질의 생성량이 수득 될 때까지 계속될 수 있으며, 구체적으로는 1 시간 내지 100 시간일 수 있으나 이에 제한되지 않는다.The temperature of the culture may be 25 °C to 40 °C, more specifically 28 °C to 37 °C, but is not limited thereto. The culturing period may be continued until a desired production amount of a useful substance is obtained, and specifically may be 1 hour to 100 hours, but is not limited thereto.
본 발명의 배양에 의하여 생산된 재조합 단백질은 배지 중으로 분비되거나 세포 내에 잔류할 수 있다.The recombinant protein produced by the culture of the present invention may be secreted into the medium or remain in the cell.
본 발명의 방법은, 본 발명의 효모 변이주를 준비하는 단계, 상기 균주를 배양하기 위한 배지를 준비하는 단계, 또는 이들의 조합(순서에 무관, in any order)을, 예를 들어, 상기 배양하는 단계 이전에, 추가로 포함할 수 있다.The method of the present invention includes the step of preparing the yeast mutant strain of the present invention, the step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, the culturing Prior to the step, it may further include.
본 발명의 방법은 상기 b) 단계 이후에 추가적인 공정을 더 포함할 수 있다. 상기 추가 공정은 상기 방법에 의해 생산된 재조합 단백질의 용도에 따라 적절히 선택될 수 있으며, 일예로, 상기 배양에 따른 배지(배양이 수행된 배지) 또는 본 발명의 효모 변이주로부터 재조합 단백질을 회수하는 단계를 추가로 포함할 수 있다. 상기 회수하는 단계는 상기 배양하는 단계 이후에 추가로 포함될 수 있다.The method of the present invention may further include an additional process after step b). The additional step may be appropriately selected depending on the use of the recombinant protein produced by the method, and for example, recovering the recombinant protein from the culture medium (culture medium) or the yeast mutant strain of the present invention. may additionally include. The recovering step may be further included after the culturing step.
상기 회수는 본 발명의 미생물의 배양 방법, 예를 들어 회분식, 연속식 또는 유가식 배양 방법 등에 따라 당해 기술 분야에 공지된 적합한 방법을 이용하여 목적하는 재조합 단백질을 수집(collect)하는 것일 수 있다. 예를 들어, 원심분리, 여과, 결정화 단백질 침전제에 의한 처리(염석법), 추출, 초음파 파쇄, 한외여과, 투석법, 분자체 크로마토그래피(겔여과), 흡착크로마토그래피, 이온교환 크로마토그래피, 친화도 크로마토그래피 등의 각종 크로마토그래피, HPLC 또는 이들의 방법을 조합하여 사용될 수 있으며, 당해 분야에 공지된 적합한 방법을 이용하여 배지 또는 미생물로부터 목적하는 재조합 단백질을 회수할 수 있다.The recovery may be to collect the desired recombinant protein using a suitable method known in the art according to the culture method of the microorganism of the present invention, for example, a batch, continuous or fed-batch culture method. For example, centrifugation, filtration, treatment with a precipitating agent for crystallized proteins (salting out method), extraction, sonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity Various types of chromatography such as chromatography, HPLC, or a combination thereof may be used, and a desired recombinant protein may be recovered from a medium or microorganism using a suitable method known in the art.
또한, 상기 회수는 상기 배양 단계 이후 재조합 단백질을 상기 균주, 이의 건조물, 추출물, 배양물, 파쇄물 중에서 선택된 하나 이상의 물질로부터 회수하는 것일 수 있다.In addition, the recovery may be to recover the recombinant protein from at least one material selected from the strain, its dried product, extract, cultured product, and lysate after the culturing step.
본 발명에서 "배양물" 은 본 발명의 균주를 배양하여 수득한 결과물일 수 있다. 예를 들어 균주를 배지에 배양하여 영양분을 섭취하고 물질대사를 통해 생겨난 부산물이 포함된 배지 일 수 있으며, 상기 배지의 희석액이나, 농축액, 상기 배지를 건조하여 얻은 건조물, 상기 배지의 조정제물이나 정제물 또는 이들의 혼합물 등, 상기 배지를 이용하여 형성 가능한 모든 제형의 배양물을 포함한다. 본 발명의 균주의 배양물은 상기 균주를 포함하거나 포함하지 않을 수 있다. 상기 배양에 대해서는 전술한 바와 같다.In the present invention, "culture" may be a result obtained by culturing the strain of the present invention. For example, it may be a medium containing nutrients obtained by culturing strains in a medium and by-products generated through metabolism, a dilution or concentrate of the medium, a dried product obtained by drying the medium, and an adjusted product or purification of the medium. It includes cultures of all formulations that can be formed using the medium, such as water or a mixture thereof. A culture of a strain of the present invention may or may not include the strain. The culture was as described above.
본 발명에서 "파쇄물"은 상기 균주 또는 이의 배양물을 파쇄 또는 용균하여 수득한 결과물, 또는 상기 파쇄물을 원심분리하여 얻어진 상등액일 수 있다.In the present invention, "lysate" may be a result obtained by crushing or lysing the strain or its culture, or a supernatant obtained by centrifuging the lysate.
상기 배양물, 파쇄물, 이들의 상등액 및 이들의 분획물 역시 본 발명 범위에 포함된다. 상기 방법은 상기 회수 단계 이전, 혹은 동시에 균주를 용균시키는 단계를 추가로 포함할 수 있다. 균주의 용균은 본 발명이 속하는 기술 분야에서 통상적으로 사용되는 방법, 예를 들어, 용균용 완충용액, 소니케이터, 열 처리 및 후렌치 프레서 등에 의해 실시할 수 있다. 또한 상기 용균 단계는 세포벽 분해 효소, 핵산 분해 효소, 핵산 전이 효소, 단백질 분해 효소 등 효소반응을 포함할 수 있으나 이에 제한되지 않는다.The cultures, lysates, supernatants thereof, and fractions thereof are also included in the scope of the present invention. The method may further include a step of lysing the strain before or simultaneously with the recovery step. Lysis of the strain may be performed by methods commonly used in the art to which the present invention pertains, for example, a lysis buffer solution, a sonicator, heat treatment, and a French press. In addition, the lysis step may include, but is not limited to, enzyme reactions such as cell wall degrading enzymes, nucleic acid degrading enzymes, nucleic acid transferases, and proteolytic enzymes.
본 발명의 목적상, 상기 재조합 단백질 제조방법을 통해 재조합 단백질이 고함량으로 포함된 건조 효모(Dry yeast), 효모 추출물(yeast extract), 효모추출물 혼합 분말(yeast extract mix powder), 순수 정제된 재조합 단백질(pure glutathione)을 제조할 수 있으나 이에 제한되지 않으며, 목적하는 제품에 따라 적절하게 제조될 수 있다.For the purpose of the present invention, dry yeast containing a high content of recombinant protein through the above recombinant protein production method, yeast extract, yeast extract mix powder, pure purified recombinant Protein (pure glutathione) can be produced, but is not limited thereto, and may be appropriately prepared according to the desired product.
본 발명에서 건조 효모(dry yeast)는 "균주 건조물" 등의 용어와 교환적으로 사용될 수 있다. 상기 건조 효모는 재조합 단백질을 축적한 효모 균체를 건조시켜 제조할 수 있으며, 구체적으로 사료용 조성물, 식품용 조성물 등에 포함될 수 있으나, 이에 제한되지 않는다.In the present invention, dry yeast (dry yeast) may be used interchangeably with terms such as "dry strain". The dry yeast may be prepared by drying the yeast cells in which the recombinant protein is accumulated, and may be specifically included in a composition for feed, a composition for food, etc., but is not limited thereto.
본 발명에서 효모 추출물(yeast extract)은 "균주 추출물" 등의 용어와 상호 교환적으로 사용될 수 있다. 상기 균주 추출물은, 상기 균주의 균체에서 세포벽을 분리하고 남은 물질을 의미할 수 있다. 구체적으로, 균체를 용균시켜 수득한 성분에서 세포벽을 제외한 나머지 성분을 의미할 수 있다. 상기 균주 추출물은 재조합 단백질을 포함하며, 재조합 단백질 외의 성분으로는 단백질, 탄수화물, 핵산, 섬유질 중 하나 이상의 성분이 포함되어 있을 수 있으나 이에 제한되지 않는다.In the present invention, yeast extract (yeast extract) may be used interchangeably with terms such as "strain extract". The strain extract may refer to a material remaining after separating cell walls from cells of the strain. Specifically, it may refer to components obtained by lysing cells except for cell walls. The strain extract includes a recombinant protein, and components other than the recombinant protein may include, but are not limited to, at least one of proteins, carbohydrates, nucleic acids, and fibers.
본 발명의 방법은 상기 b) 단계 이후에 재조합 단백질을 정제하는 단계를 추가로 포함할 수 있다. 상기 정제는 당해 기술분야에 공지된 적합한 방법을 이용하여 수행할 수 있다. 일예로, 본 발명의 재조합 단백질 생산 방법이 회수 단계와 정제 단계를 모두 포함하는 경우, 상기 회수 단계와 정제 단계는 순서에 상관없이 연속적 또는 비연속적으로 수행되거나, 동시에 또는 하나의 단계로 통합되어 수행될 수 있으나, 이에 제한되는 것은 아니다.The method of the present invention may further include purifying the recombinant protein after step b). The purification may be performed using suitable methods known in the art. For example, when the recombinant protein production method of the present invention includes both a recovery step and a purification step, the recovery step and the purification step are performed continuously or discontinuously regardless of order, or performed simultaneously or integrated into one step. It may be, but is not limited thereto.
본 발명에 따른 갈락토스 이용에 관여하는 모든 유전자 결손된 변이주는 갈락토스 사용 없이도 종래 균주보다 재조합 단백질 발현율과 세포 성장이 현저히 우수하므로 저렴한 비용으로 재조합 단백질을 대량생산할 수 있는 바, 산업 용도의 재조합 단백질 발현 시스템 구축에 적용할 수 있다.All gene-defective mutants involved in the use of galactose according to the present invention have significantly better recombinant protein expression rates and cell growth than conventional strains even without the use of galactose, so that recombinant proteins can be mass-produced at low cost. Recombinant protein expression system for industrial use can be applied to construction.
도 1은 갈락토스 이용 관련 유전자(GAL 유전자) 결손에 사용된 pCAS 벡터의 구조를 나타낸 것이다.
도 2는 CRISPR-CAS9 시스템을 이용하여 GAL1, GAL10, GAL7 유전자를 동시에 결손시키는 과정을 나타낸 모식도이다.
도 3은 콜로니 PCR을 이용하여 GAL1, GAL10, GAL7 유전자가 동시에 결손된 것을 확인한 도이다.
도 4는 CRISPR-CAS9 시스템을 이용하여 GAL2 유전자를 결손시키는 과정을 나타낸 도이다.
도 5는 콜로니 PCR을 이용하여 GAL2 유전자가 결손된 것을 확인한 도이다.
도 6은 CalB14를 발현하는 6종의 재조합 균주의 성장을 비교한 도이다.
도 7은 CalB14를 발현하는 6종의 재조합 균주의 시간별 CalB14 발현량을 SDS-PAGE를 이용하여 확인한 도이다.
도 8은 CalB14를 발현하는 6종의 재조합 균주의 시간별로 발현된 CalB14의 활성을 확인한 도이다.
도 9는 CalB14를 발현하는 gal80 균주와 allgal 균주의 성장(A) 및 단백질 발현량(B)을 비교한 도이다.
도 10은 EGF를 발현하는 gal80 균주와 allgal 균주의 성장(A) 및 단백질 발현량(B)을 비교한 도이다.Figure 1 shows the structure of the pCAS vector used for galactose utilization-related gene (GAL gene) deficiency.
2 is a schematic diagram showing a process of simultaneously deleting GAL1, GAL10, and GAL7 genes using the CRISPR-CAS9 system.
3 is a diagram confirming simultaneous deletion of GAL1, GAL10, and GAL7 genes using colony PCR.
4 is a diagram showing the process of deleting the GAL2 gene using the CRISPR-CAS9 system.
5 is a diagram confirming that the GAL2 gene is deleted using colony PCR.
Figure 6 is a diagram comparing the growth of six recombinant strains expressing CalB14.
7 is a diagram confirming the expression level of CalB14 over time in six recombinant strains expressing CalB14 using SDS-PAGE.
8 is a diagram confirming the activity of CalB14 expressed over time in six recombinant strains expressing CalB14.
Figure 9 is a comparison of the growth (A) and protein expression level (B) of the gal80 strain and allgal strain expressing CalB14.
Figure 10 is a diagram comparing the growth (A) and protein expression level (B) of the gal80 strain and allgal strain expressing EGF.
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
실시예 1. 갈락토스 이용 관련 유전자 결손 균주 제작Example 1. Production of gene-deficient strains related to the use of galactose
갈락토스 이용 관련 유전자(GAL 유전자)들을 결손한 변이주 제작을 위해 모균주로 사카로마이세스 세레비지에(Saccharomyces cerevisiae) 2805 균주(MATα pep4::HIS3 prb1 can1 his3-200 ura3-52)(Sohn 등, J. Microbiol. Biotechnol. 1, 266, 1991)와 GAL80 유전자(서열번호 1)의 프로모터 및 코딩서열 (Coding sequence)의 일부분이 결손된 사카로마이세스 세레비지에 2805△gal80[Whang Jake 등, Process Biochemistry, 44, 1190-1192(2009)] 균주를 사용하였다. Saccharomyces cerevisiae 2805 strain (MATα pep4::HIS3 prb1 can1 his3-200 ura3-52) (Sohn et al., J. Microbiol. Biotechnol. 1, 266, 1991) and Saccharomyces cerevisiae 2805Δgal80 [Whang Jake et al., Process Biochemistry, 44, 1190-1192 (2009)] strain was used.
구체적으로, GAL1, GAL10, GAL7, GAL2 유전자가 모두 결손된 변이주를 제작하기 위하여 3세대 유전자 가위 기술인 CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)-CAS9 기술을 이용하여 GAL 변이주를 제작하였다. 사카로마이세스 세레비지에 CRISPR-CAS9 벡터는 Addgene사에서 pCAS 벡터를 구입하여 사용하였다. pCAS 벡터는 DNA 서열상에서 지정된 위치를 자르는 DNase인 CAS9 효소와 CAS9을 지정된 위치로 유도하는 유도 RNA(gRNA)가 하나의 벡터에서 동시에 발현되는 구조이다(도 1).Specifically, GAL mutants were prepared using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-CAS9 technology, a third-generation genetic scissors technology, to create mutants lacking all of the GAL1, GAL10, GAL7, and GAL2 genes. Saccharomyces cerevisiae CRISPR-CAS9 vector was used by purchasing a pCAS vector from Addgene. The pCAS vector is a structure in which a CAS9 enzyme, which is a DNase that cuts a designated site on a DNA sequence, and a guide RNA (gRNA) that guides CAS9 to a designated site are simultaneously expressed in one vector (FIG. 1).
목적하는 위치에서 CAS9이 DNA를 절단할 수 있도록 유도하기 위해서는 gRNA의 특정 부위에 존재하는 연속되는 20개의 염기를 목적하는 위치 서열로 변경해야 한다. GAL7 유전자의 ORF(open reading frame) 내에서 자르기 위한 gRNA 서열은 GATTGTAACGTCTATGGGAA(서열번호 8)로 선정하였고, GAL2 유전자의 프로모터 부위를 자르기 위한 gRNA 서열은 CAATTCGGAAAGCTTCCTTC(서열번호 9)로 선정하였다. 이후, PCR을 이용하여 선정된 각 서열을 함유하는 gRNA를 발현하는 벡터를 각각 제작하고 pCAS-gGAL7, pCAS-gGAL2로 명명하였다(도 1). 각 유전자의 gRNA용 목적 부위 선정은 CRISPRdirect 프로그램(https://crispr.dbcls.jp/)을 이용하여 결정하였다.In order to induce CAS9 to cleave DNA at the desired location, 20 consecutive bases at a specific site of the gRNA must be changed to the sequence at the desired location. The gRNA sequence for cutting within the ORF (open reading frame) of the GAL7 gene was selected as GATTGTAACGTCTATGGGAA (SEQ ID NO: 8), and the gRNA sequence for cutting the promoter region of the GAL2 gene was selected as CAATTCGGAAAGCTTCCTTC (SEQ ID NO: 9). Thereafter, vectors expressing gRNAs containing the selected sequences were constructed using PCR, respectively, and named pCAS-gGAL7 and pCAS-gGAL2 (FIG. 1). Selection of the target site for gRNA of each gene was determined using the CRISPRdirect program (https://crispr.dbcls.jp/).
GAL1, GAL10 및 GAL7 유전자는 2번 염색체에 인접해서 존재하고 있기 때문에 한번에 상기 3개 유전자의 결손을 시도하였다. pCAS-gGAL7 벡터와 함께 사용할 결손 카세트(disruption cassette)를 제작하기 위하여 사카로마이세스 세레비지에 2805 게놈 유전자를 주형으로 H978 프라이머(서열번호 10) 및 H979 프라이머(서열번호 11)를 이용하여 GAL1 유전자 400bp를 증폭하였다. H980 프라이머(서열번호 12) 및 H981 프라이머(서열번호 13)를 이용하여 동일한 방법으로 GAL7 유전자 400bp를 증폭하였다. H979 프라이머(서열번호 11)와 H980 프라이머(서열번호 12)는 서로 상보적인 서열을 포함하고 있기 때문에 증폭한 각각의 PCR 산물은 H978 프라이머(서열번호 10) 및 H981 프라이머(서열번호 13)를 이용하여 overlap extension PCR을 수행하여 하나의 유전자 단편으로 연결하였다(도 2). Since the GAL1, GAL10, and GAL7 genes exist adjacent to chromosome 2, deletion of the three genes was attempted at once. To construct a disruption cassette to be used with the pCAS-gGAL7 vector, the Saccharomyces cerevisiae 2805 genomic gene was used as a template and the H978 primer (SEQ ID NO: 10) and the H979 primer (SEQ ID NO: 11) were used to generate the GAL1 gene. 400bp was amplified. Using the H980 primer (SEQ ID NO: 12) and the H981 primer (SEQ ID NO: 13), 400 bp of the GAL7 gene was amplified in the same manner. Since the H979 primer (SEQ ID NO: 11) and the H980 primer (SEQ ID NO: 12) contain complementary sequences, each PCR product amplified was obtained using the H978 primer (SEQ ID NO: 10) and the H981 primer (SEQ ID NO: 13). Overlap extension PCR was performed to link them into one gene fragment (FIG. 2).
제작된 결손 카세트와 pCAS-gGAL7 벡터를 사카로마이세스 세레비지에 균주에 동시에 도입하여 CAS9에 의한 GAL7 유전자 위치에서의 절단을 유도하고, 절단된 염색체의 말단 부위에 결손 카세트를 상동유전자 재조합으로 연결하였다. 결손 카세트 도입 후 GAL1 유전자(서열번호 2)의 프로모터 및 코딩서열(Coding sequence) 일부, GAL10 유전자(서열번호 3)의 프로모터 및 코딩서열 일부와, GAL7 유전자(서열번호 4)의 프로모터 및 코딩서열 일부를 포함하는 총 2.8kb 길이의 염기서열(서열번호 6)이 결손되었다(도 2). 이때 사용한 형질전환 방법은 리튬아세테이트 방법[Gietz RD 등 Yeast, 11, 355-360(1995)]을 이용하였으며 형질전환체는 항생제 G418이 100νg/ml농도로 포함되어있는 YPD 고체배지(효모 추출물 1%, 펩톤 2%, 포도당 2%, 아가 2%)에서 선별하였다. 30℃에서 2일간 배양 후 형성된 형질전환체에서 무작위적으로 선택한 8개의 콜로니를 리티카제(lyticase)가 2mg/ml 농도로 포함된 10mM 인산나트륨 완충액(Sodium phosphate, pH 8)에 현탁하고 37℃에서 30분간 반응시켜 세포벽을 분해하였으며, 단백질 분해효소를 1mg/ml이 되도록 첨가하여 50℃에서 10분간 반응한 후 H978(서열번호 10) 프라이머 및 H981 프라이머(서열번호 13)를 이용하여 PCR을 수행하여 GAL1, GAL10 및 GAL7 유전자가 모두 결손된 균주를 확인하였다(도 3). 야생형은 PCR 하면 3.6kb로 증폭되는 반면 GAL1, GAL10 및 GAL7 유전자가 동시에 모두 결손된 변이주는 0.8kb의 크기로 증폭되기 때문에 유전자 결실을 쉽게 확인할 수 있다. 사카로마이세스 세레비지에 2805 균주의 GAL1, GAL10 및 GAL7 유전자 결손을 시도했을 경우(2805△gal1△gal10△gal7) 확인한 8종의 콜로니 모두에서 유전자 결실이 확인되었으며, 2805△gal80 균주의 GAL1, GAL10 및 GAL7 유전자 결손을 시도했을 경우(2805△gal80△gal1△gal10△gal7) 형질전환된 8개의 콜로니 중 5개에서 유전자 결실이 확인되었다(도 3). 이에 따라, 한번의 형질전환으로 GAL1, GAL10 및 GAL7 유전자가 동시에 결손된 균주를 제작하였음을 확인하였다. 형질전환체에 도입된 pCAS-gGAL7 벡터는 G418 항생제를 포함하지 않는 YPD 배지에 배양하여 자연적으로 pCAS-gGAL7 벡터가 제거된 균주를 선별하였다.The prepared defective cassette and the pCAS-gGAL7 vector were simultaneously introduced into a Saccharomyces cerevisiae strain to induce cleavage at the location of the GAL7 gene by CAS9, and link the defective cassette to the end of the chromosome by homologous recombination. did After introduction of the defective cassette, part of the promoter and coding sequence of the GAL1 gene (SEQ ID NO: 2), part of the promoter and coding sequence of the GAL10 gene (SEQ ID NO: 3), and part of the promoter and coding sequence of the GAL7 gene (SEQ ID NO: 4) The base sequence (SEQ ID NO: 6) with a total length of 2.8 kb including was deleted (FIG. 2). The transformation method used at this time was the lithium acetate method [Gietz RD et al. Yeast, 11, 355-360 (1995)], and the transformants were YPD solid medium containing the antibiotic G418 at a concentration of 100 νg / ml (yeast extract 1% , peptone 2%, glucose 2%, agar 2%). Eight colonies randomly selected from the transformants formed after culturing at 30 ° C for 2 days were suspended in 10 mM sodium phosphate buffer (Sodium phosphate, pH 8) containing lyticase at a concentration of 2 mg / ml, and then incubated at 37 ° C. The cell wall was degraded by reacting for 30 minutes at 1 mg/ml of proteolytic enzyme, reacted at 50 ° C for 10 minutes, and then PCR was performed using H978 (SEQ ID NO: 10) primers and H981 primers (SEQ ID NO: 13). Thus, strains lacking all of the GAL1, GAL10, and GAL7 genes were confirmed (FIG. 3). The wild type is amplified to 3.6 kb by PCR, whereas the mutant in which GAL1, GAL10 and GAL7 genes are all deleted at the same time is amplified to 0.8 kb in size, so gene deletion can be easily confirmed. When GAL1, GAL10, and GAL7 gene deletion of Saccharomyces cerevisiae strain 2805 was attempted (2805Δgal1Δgal10Δgal7), gene deletion was confirmed in all eight colonies identified, and GAL1, GAL10 and GAL7 of strain 2805Δgal80 When GAL10 and GAL7 gene deletion was attempted (2805Δgal80Δgal1Δgal10Δgal7), gene deletion was confirmed in 5 out of 8 transformed colonies (FIG. 3). Accordingly, it was confirmed that strains in which GAL1, GAL10, and GAL7 genes were simultaneously deleted were prepared through one-time transformation. The pCAS-gGAL7 vector introduced into the transformants was cultured in YPD medium without the G418 antibiotic, and strains from which the pCAS-gGAL7 vector was naturally removed were selected.
추가로, 12번 염색체에 존재하는 GAL2 유전자를 결손시키기 위해, 상기 방법과 동일하게 H984 프라이머(서열번호 14) 및 H985 프라이머(서열번호 15)를 이용하여 GAL2 유전자의 프로모터 부위 400bp를 증폭하고 H986 프라이머(서열번호 16) 및 H987 프라이머(서열번호 17)를 이용하여 GAL2 유전자의 ORF 중 일부에 해당하는 400bp의 염기를 증폭시키고, H984 프라이머(서열번호 14) 및 H987 프라이머(서열번호 17)를 이용한 overlap extension PCR로 연결하여 결손 카세트를 제작하였다(도 4). 제작된 결손 카세트와 pCAS-gGAL2 벡터를 2805△gal80△gal1△gal10△gal7 균주에 형질전환하여 형질전환체를 선별하였으며 무작위로 선택한 8개의 콜로니에 대해 H984 프라이머(서열번호 14) 및 H987 프라이머(서열번호 17)를 이용한 PCR을 수행하여 GAL2 유전자(서열번호 5)의 프로모터 및 코딩서열 일부(서열번호 7)의 결손 여부를 확인하였다(도 5). Additionally, in order to delete the GAL2 gene present on
야생형이 2.6kb로 증폭되고 GAL2 유전자가 결손된 변이주는 0.8kb의 크기로 증폭되었는지를 확인한 결과, 모든 콜로니가 0.8kb로 증폭되었기 때문에 GAL2 유전자가 정확히 결손되었음을 알 수 있다. 형질전환체에 도입된 pCAS-gGAL2 벡터는 G418 항생제를 포함하지 않는 YPD 배지에 배양하여 자연적으로 벡터가 제거된 균주를 선별하였다.As a result of confirming whether the wild type was amplified to 2.6 kb and the GAL2 gene-deleted mutant was amplified to 0.8 kb, it was confirmed that the GAL2 gene was correctly deleted because all colonies were amplified to 0.8 kb. The pCAS-gGAL2 vector introduced into the transformant was cultured in YPD medium without the G418 antibiotic to select a strain from which the vector was naturally removed.
본 실시예에서 사용된 프라이머는 하기 표 1에 나타내었다.The primers used in this Example are shown in Table 1 below.
또한, 본 실시예에서 제작된 변이 균주와 비교 실험에 사용한 균주의 유전자형은 하기 표 2에 나타내었다.In addition, the genotypes of the mutant strains prepared in this Example and the strains used in the comparative experiment are shown in Table 2 below.
실시예 2. 갈락토스 이용 관련 유전자 결손 균주의 성장 및 재조합 단백질 발현 비교Example 2. Comparison of growth and recombinant protein expression of strains with gene deletion related to galactose utilization
상기 실시예 1에서 제작된 GAL 유전자 결손 균주의 성장과 재조합 단백질 발현량을 비교하기 위해, 상기 표 1의 균주 중 2805△gal80,1,10,7 균주를 제외한 5개 균주에 캔디다 앤탁티카(Candida antartica) 리파제 B 유전자(CalB)의 변이체인 CalB14(한국 등록특허 제10-0697310호)를 포함하는 CalB14 분비 발현 벡터(YGa-T3CalB14)를 도입하여 발현을 유도하였다. YGa-T3CalB14 벡터는 GAL10 프로모터를 이용하여 CalB14를 고효율로 분비 발현하고, YG-T3CalB14 벡터는 구성적 프로모터인 TDH3(glyceraldehyde 3-phosphate dehydrogenase) 프로모터를 이용하여 CalB14를 분비 발현하였다. YGa-T3CalB14 벡터로 형질전환된 2805 균주는 GAL10 프로모터의 발현을 유도하기 위하여 YPDG 배지(포도당 1%, 갈락토스 1%)에서 배양하였고 YGa-T3CalB14 벡터로 형질전환된 2805△gal1 균주와 2805△gal1,10,7 균주는 GAL10 프로모터의 발현을 유도하기 위하여 갈락토스가 최소량 포함된 YPDG 배지(포도당 1%, 갈락토스 0.05%)에서 배양하였다. YG-T3CalB14 벡터로 형질전환된 2805 균주와 나머지 균주는 모두 YPD 배지(포도당 2%)에서 배양하였다. In order to compare the growth and recombinant protein expression levels of the GAL gene-defective strains prepared in Example 1, five strains except 2805Δgal80,1,10,7 strains among the strains in Table 1 were tested for Candida Expression was induced by introducing a CalB14 secretion expression vector (YGa-T3CalB14) containing CalB14 (Korean Patent Registration No. 10-0697310), which is a variant of the antartica lipase B gene (CalB). The YGa-T3CalB14 vector secreted and expressed CalB14 with high efficiency using the GAL10 promoter, and the YG-T3CalB14 vector secreted and expressed CalB14 using the TDH3 (glyceraldehyde 3-phosphate dehydrogenase) promoter, a constitutive promoter. The 2805 strain transformed with the YGa-T3CalB14 vector was cultured in YPDG medium (glucose 1%, galactose 1%) to induce expression of the GAL10 promoter. 10,7 strain was cultured in YPDG medium (glucose 1%, galactose 0.05%) containing a minimum amount of galactose to induce expression of the GAL10 promoter. The 2805 strain transformed with the YG-T3CalB14 vector and the remaining strains were all cultured in YPD medium (2% glucose).
본 실시예에서 사용한 6종 균주별 발현 벡터 및 배지는 하기 표 3에 나타내었다.Expression vectors and media for each of the six strains used in this Example are shown in Table 3 below.
상기 6종의 균주를 YPD 액체배지에서 밤새 배양한 후, 표 2에 표시한 배지를 50ml로 포함하는 각 플라스크에서 OD600을 측정하며 1.0이 되도록 접종하였다. 이후, 30℃에서 48시간 동안 각 균주를 진탕배양하면서 흡광도를 이용하여 성장을 측정하였다.After culturing the above six strains overnight in YPD liquid medium, the OD 600 was measured in each flask containing 50 ml of the medium shown in Table 2 and inoculated so as to be 1.0. Thereafter, growth was measured using absorbance while shaking culture of each strain at 30 ° C. for 48 hours.
그 결과, 도 6과 같이 GAL10 프로모터를 포함하는 YGa-T3CalB14 벡터로 형질전환된 gal1 변이주, YGa-T3CalB14 벡터로 형질전환된 gal1,10,7 변이주 및 TDH3 프로모터를 포함하는 YG-T3CalB14 벡터로 형질전환된 야생형 균주는 YGa-T3CalB14 벡터로 형질전환된 야생형 균주보다 성장이 느린 것을 확인하였다. 또한, YGa-T3CalB14 벡터로 형질전환된 gal80 균주와 allgal 균주는 YGa-T3CalB14 벡터로 형질전환된 야생형 균주보다 성장이 약 10-15% 더 우수하였다. As a result, as shown in FIG. 6, the gal1 mutant strain transformed with the YGa-T3CalB14 vector containing the GAL10 promoter, the gal1,10,7 mutant strain transformed with the YGa-T3CalB14 vector, and the YG-T3CalB14 vector containing the TDH3 promoter were transformed. It was confirmed that the growth of the wild-type strain was slower than that of the wild-type strain transformed with the YGa-T3CalB14 vector. In addition, the gal80 and allgal strains transformed with the YGa-T3CalB14 vector showed about 10-15% better growth than the wild-type strain transformed with the YGa-T3CalB14 vector.
또한, 시간별로 분비된 CalB14의 양을 SDS-PAGE로 비교한 결과, 균주 성장과 비례하는 발현량을 나타내었다(도 7).In addition, as a result of comparing the amount of CalB14 secreted over time by SDS-PAGE, the expression level was proportional to the growth of the strain (FIG. 7).
다음으로, 각 균주로부터 발현되어 배양액에 포함된 리파제의 활성을 발색시약인 p-니트로페닐 팔미테이트(p-nitrophenyl palmitate, pNPP)을 이용하여 측정하였다. 구체적으로, 10 μl의 10 mM pNPP, 40 μl 에틸 알콜 및 950 μl 50mM pH 7.5 트리스 완충액이 조합된 반응액 1 ml에 적절히 희석된 20 μl의 효소 용액을 넣고 10분간 반응 후 405nm에서 흡광도를 측정하여 확인하였다. 리파제 활성 1 unit은 1분당 1 μmole의 pNP기를 유리시키는 효소의 활성으로 정의하였다.Next, the activity of lipase expressed from each strain and included in the culture medium was measured using p-nitrophenyl palmitate (pNPP), a color developing reagent. Specifically, 10 μl of 10 mM pNPP, 40 μl of ethyl alcohol and 950 μl of 50 mM pH 7.5 Tris buffer were mixed with 20 μl of an enzyme solution appropriately diluted in 1 ml of the reaction solution, reacted for 10 minutes, and then measured the absorbance at 405 nm. Confirmed. One unit of lipase activity was defined as the activity of an enzyme that liberates 1 µmole of pNP group per minute.
그 결과, gal80 균주와 allgal 균주의 효소 활성이 가장 높음을 확인하였다(도 8). As a result, it was confirmed that the gal80 strain and the allgal strain had the highest enzyme activity (FIG. 8).
이에 따라, 구성적 프로모터인 TDH3 프로모터를 이용한 경우 GAL10 프로모터를 이용한 경우에 비하여 균주 성장과 재조합 단백질 발현량이 적고, gal1 변이주를 사용하여 최소량의 갈락토스로 발현하는 경우에도 야생형 균주에서 GAL10 프로모터를 이용해서 발현하는 경우에 비하여 균주 성장과 재조합 단백질 발현량이 적음을 확인하였다. 또한, gal1 변이주에 GAL10 및 GAL7 유전자를 추가 결손시켜도 균주의 성장이나 재조합 단백질 발현에 영향이 없는 것을 확인하였다. Accordingly, when the TDH3 promoter, which is a constitutive promoter, is used, the strain growth and recombinant protein expression are lower than when the GAL10 promoter is used. It was confirmed that the growth of the strain and the expression of the recombinant protein were less than in the case of In addition, it was confirmed that the growth of the strain or the recombinant protein expression was not affected even when the GAL10 and GAL7 genes were additionally deleted in the gal1 mutant strain.
결론적으로, 사카로마이세스 세레비지에 균주에서 재조합 단백질을 발현하는 경우 GAL10 프로모터를 포함하는 재조합 단백질 발현 벡터를 2805△gal80 균주 또는 2805△allgal 균주에 형질전환하여 재조합 단백질을 발현시키는 것이 균주 성장과 재조합 단백질 발현에 있어 최적 조건임을 확인하였다. In conclusion, when expressing a recombinant protein in a Saccharomyces cerevisiae strain, transforming the 2805Δgal80 strain or 2805Δallgal strain with a recombinant protein expression vector containing the GAL10 promoter to express the recombinant protein results in strain growth and It was confirmed that this was an optimal condition for recombinant protein expression.
실시예 3. 유가식 배양을 통한 갈락토스 이용 관련 유전자 결손 균주의 성장 및 재조합 단백질 발현 비교Example 3. Comparison of growth and recombinant protein expression of galactose utilization-related gene-deficient strains through fed-batch culture
상기 실시예 2에서 확인한 바와 같이 GAL10 프로모터를 사용하고, 2805△gal80 균주 또는 2805△allgal 균주를 YPD 배지에서 배양하여 재조합 단백질을 발현한 경우 균주 성장과 재조합 단백질 발현량이 가장 우수함을 확인하였으나 플라스크 배양으로 이 두 균주의 차이를 명확히 확인하기는 어려웠기 때문에 YGa-T3CalB14 벡터로 형질전환된 두 균주를 5L 발효조를 이용한 유가식 배양을 통하여 균주 성장과 재조합 단백질 발현량을 비교하였다. As confirmed in Example 2, when the GAL10 promoter was used and the 2805Δgal80 strain or the 2805Δallgal strain was cultured in YPD medium to express the recombinant protein, it was confirmed that the growth of the strain and the expression of the recombinant protein were the best. Since it was difficult to clearly identify the difference between these two strains, the two strains transformed with the YGa-T3CalB14 vector were compared in fed-batch culture using a 5L fermentor in terms of strain growth and recombinant protein expression.
구체적으로, 시드(Seed) 배양은 50ml 최소 액체배지(0.67% 아미노산이 결여된 효모 질소원 기질, 0.5% 카사미노산, 2% 포도당)에서 24시간 동안 배양 후 다시 200ml YPD 배지에서 배양하여 활성화시키고, 본 배양액에 접종하여 30℃에서 48시간 동안 배양하였다. 세포 성장 속도에 따라 유가식 배양 배지(15% 효모 추출물, 30% 포도당)를 추가 공급하였다. 배양 시간별로 배양액의 흡광도를 측정하였고 Bradford 단백질 정량법을 이용하여 배양 상등액에 포함된 전체 단백질을 정량하였다. Specifically, the seed culture was cultured in 50ml minimal liquid medium (yeast nitrogen source substrate lacking 0.67% amino acids, 0.5% casamino acid, 2% glucose) for 24 hours, and then cultured in 200ml YPD medium again to activate. Inoculated into the culture medium and incubated at 30 ℃ for 48 hours. Fed-batch culture medium (15% yeast extract, 30% glucose) was additionally supplied according to the cell growth rate. The absorbance of the culture solution was measured for each incubation time, and the total protein contained in the culture supernatant was quantified using the Bradford protein quantification method.
그 결과, 2805△gal80 균주는 48시간 동안 발효 후 OD600 143까지 성장한 반면 2805△allgal 균주는 이보다 21% 증가된 172까지 성장하였으며(도 9A), 분비 생산된 단백질의 양은 2805△allgal 균주가 0.63g/L로 2805△gal80 균주(0.56g/L)에 비해 12% 증가하여(도 9B) 유가식 배양에서 두 균주간의 차이가 확연하게 나타남을 확인하였다.As a result, the 2805Δgal80 strain grew to an OD 600 of 143 after fermentation for 48 hours, whereas the 2805Δallgal strain grew to 172, a 21% increase (Fig. 9A), and the amount of secreted protein produced by the 2805Δallgal strain was 0.63. It was confirmed that g/L increased by 12% compared to the 2805Δgal80 strain (0.56 g/L) (FIG. 9B), showing a clear difference between the two strains in fed-batch culture.
다음으로, 2805△allgal 균주의 성장 및 재조합 단백질 생산성 향상 효과가 목적 단백질에 따라 영향을 받는지 확인하기 위하여 동일 균주에 인간 상피세포 성장인자(human Epidermal growth factor, EGF) 발현 벡터를 형질전환하여 균주 성장과 재조합 단백질 발현량을 비교하였다. EGF 발현 벡터는 YGa-T3CalB14 벡터와 유사한 골격으로 구성하였으며, GAL10 프로모터와 Mating factor 분비 시그널을 이용하여 EGF를 세포 밖으로 분비 발현하도록 구성되었다(YGa-T1EGF). Next, in order to determine whether the growth and recombinant protein productivity improvement effects of the 2805Δallgal strain are affected by the target protein, the same strain is transformed with a human epidermal growth factor (EGF) expression vector to grow the strain and recombinant protein expression levels were compared. The EGF expression vector has a skeleton similar to that of the YGa-T3CalB14 vector, and is configured to secrete and express EGF outside the cell using the GAL10 promoter and mating factor secretion signal (YGa-T1EGF).
그 결과, EGF를 발현하는 2805△gal80 균주는 48시간 동안 발효 후 OD600 106까지 성장한 반면 2805△allgal 균주는 이보다 38% 증가된 146까지 성장하였으며(도 10a), 분비 생산된 단백질의 양은 2805△allgal 균주가 0.59g/L로 2805△gal80 균주(0.48g/L)에 비해 22% 증가하였다(도 10b). As a result, the 2805Δgal80 strain expressing EGF grew to OD 600 of 106 after fermentation for 48 hours, whereas the 2805Δ allgal strain grew to 146, a 38% increase (Fig. 10a), and the amount of secreted protein produced was 2805Δ The allgal strain increased by 22% to 0.59g/L compared to the 2805Δgal80 strain (0.48g/L) (FIG. 10b).
결론적으로, 2805△allgal 균주는 목적 단백질의 종류와 무관하게 2805△gal80 균주보다 성장이 21-38% 개선되었으며 분비 생산되는 재조합 단백질의 양도 12-22% 증가함을 알 수 있다.In conclusion, it can be seen that the growth of the 2805Δallgal strain was improved by 21-38% compared to the 2805Δgal80 strain, and the amount of secreted and produced recombinant protein increased by 12-22%, regardless of the type of target protein.
상기 2805△allgal 균주는 부다페스트조약 하의 수탁기관인 한국생명공학연구원 생물자원센터에 2021년 5월 27일자로 기탁하여 수탁번호 KCTC 18901P 를 부여받았다.The 2805Δallgal strain was deposited on May 27, 2021 with the Korea Research Institute of Bioscience and Biotechnology, an entrusted institution under the Budapest Treaty, and was given accession number KCTC 18901P.
상기 실시예의 결과로부터, 본 발명에 따른 갈락토스 이용에 관여하는 모든 유전자(GAL)가 결손된 변이주(allgal)는 갈락토스 사용 없이도 종래 균주보다 재조합 단백질 발현율과 세포 성장이 현저히 우수하므로 저렴한 비용으로 재조합 단백질을 대량생산할 수 있음을 확인하였는 바, 산업 용도의 재조합 단백질 발현 시스템 구축에 적용할 수 있다.From the results of the above examples, the mutant strain (allgal) lacking all genes (GAL) involved in galactose utilization according to the present invention has significantly better recombinant protein expression rate and cell growth than conventional strains without using galactose, so that recombinant protein can be produced at low cost. Since it was confirmed that mass production is possible, it can be applied to constructing a recombinant protein expression system for industrial use.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 다른 당 업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변경된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be embodied in other specific forms without changing its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modified forms derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.
<110> Korea Research Institute of Bioscience and Biotechnology Cellapy Bio Inc. <120> Yeast strain in which all genes involved in galactose utilization are deleted and method for producing recombinant protein using the same <130> KPA210659-KR <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 1308 <212> DNA <213> Unknown <220> <223> GAL80 NT <400> 1 atggactaca acaagagatc ttcggtctca accgtgccta atgcagctcc cataagagtc 60 ggattcgtcg gtctcaacgc agccaaagga tgggcaatca agacacatta ccccgccata 120 ctgcaactat cgtcacaatt tcaaatcact gccttataca gtccaaaaat tgagacttct 180 attgccacca ttcagcgtct aaaattgagt aatgccactg cttttcccac tttagagtca 240 tttgcatcat cttccactat agatatgata gtgatagcta tccaagtggc cagccattat 300 gaagttgtta tgcctctctt ggaattctcc aaaaataatc cgaacctcaa gtatcttttc 360 gtagaatggg cccttgcatg ttcactagat caagccgaat ccatttataa ggctgctgct 420 gaacgtgggg ttcaaaccat catctcttta caaggtcgta aatcaccata tattttgaga 480 gcaaaagaat taatatctca aggctatatc ggcgacatta attcgatcga gattgctgga 540 aatggcggtt ggtacggcta cgaaaggcct gttaaatcac caaaatacat ctatgaaatc 600 gggaacggtg tagatctggt aaccacaaca tttggtcaca caatcgatat tttacaatac 660 atgacaagtt cgtacttttc caggataaat gcaatggttt tcaataatat tccagagcaa 720 gagctgatag atgagcgtgg taaccgattg ggccagcgag tcccaaagac agtaccggat 780 catcttttat tccaaggcac attgttaaat ggcaatgttc cagtgtcatg cagtttcaaa 840 ggtggcaaac ctaccaaaaa atttaccaaa aatttggtca ttgacattca cggtaccaag 900 ggagatttga aacttgaagg cgatgccggc ttcgcagaaa tttcaaatct ggtcctttac 960 tacagtggaa ctagagcaaa cgacttcccg ctagccaatg gacaacaagc tcctttagac 1020 ccggggtatg atgcaggtaa agaaatcatg gaagtatatc atttacgaaa ttataatgcc 1080 attgtgggta atattcatcg actgtatcaa tctatctctg acttccactt caatacaaag 1140 aaaattcctg aattaccctc acaatttgta atgcaaggtt tcgatttcga aggctttccc 1200 accttgatgg atgctctgat attacacagg ttaatcgaga gcgtttataa aagtaacatg 1260 atgggctcca cattaaacgt tagcaatatc tcgcattata gtttataa 1308 <210> 2 <211> 1587 <212> DNA <213> Unknown <220> <223> GAL1 NT <400> 2 atgactaaat ctcattcaga agaagtgatt gtacctgagt tcaattctag cgcaaaggaa 60 ttaccaagac cattggccga aaagtgcccg agcataatta agaaatttat aagcgcttat 120 gatgctaaac cggattttgt tgctagatcg cctggtagag tcaatctaat tggtgaacat 180 attgattatt gtgacttctc ggttttacct ttagctattg attttgatat gctttgcgcc 240 gtcaaagttt tgaacgagaa aaatccatcc attaccttaa taaatgctga tcccaaattt 300 gctcaaagga agttcgattt gccgttggac ggttcttatg tcacaattga tccttctgtg 360 tcggactggt ctaattactt taaatgtggt ctccatgttg ctcactcttt tctaaagaaa 420 cttgcaccgg aaaggtttgc cagtgctcct ctggccgggc tgcaagtctt ctgtgagggt 480 gatgtaccaa ctggcagtgg attgtcttct tcggccgcat tcatttgtgc cgttgcttta 540 gctgttgtta aagcgaatat gggccctggt tatcatatgt ccaagcaaaa tttaatgcgt 600 attacggtcg ttgcagaaca ttatgttggt gttaacaatg gcggtatgga tcaggctgcc 660 tctgtttgcg gtgaggaaga tcatgctcta tacgttgagt tcaaaccgca gttgaaggct 720 actccgttta aatttccgca attaaaaaac catgaaatta gctttgttat tgcgaacacc 780 cttgttgtat ctaacaagtt tgaaaccgcc ccaaccaact ataatttaag agtggtagaa 840 gtcactacag ctgcaaatgt tttagctgcc acgtacggtg ttgttttact ttctggaaaa 900 gaaggatcga gcacgaataa aggtaatcta agagatttca tgaacgttta ttatgccaga 960 tatcacaaca tttccacacc ctggaacggc gatattgaat ccggcatcga acggttaaca 1020 aagatgctag tactagttga agagtctctc gccaataaga aacagggctt tagtgttgac 1080 gatgtcgcac aatccttgaa ttgttctcgc gaagaattca caagagacta cttaacaaca 1140 tctccagtga gatttcaagt cttaaagcta tatcagaggg ctaagcatgt gtattctgaa 1200 tctttaagag tcttgaaggc tgtgaaatta atgactacag cgagctttac tgccgacgaa 1260 gactttttca agcaatttgg tgccttgatg aacgagtctc aagcttcttg cgataaactt 1320 tacgaatgtt cttgtccaga gattgacaaa atttgttcca ttgctttgtc aaatggatca 1380 tatggttccc gtttgaccgg agctggctgg ggtggttgta ctgttcactt ggttccaggg 1440 ggcccaaatg gcaacataga aaaggtaaaa gaagcccttg ccaatgagtt ctacaaggtc 1500 aagtacccta agatcactga tgctgagcta gaaaatgcta tcatcgtctc taaaccagca 1560 ttgggcagct gtctatatga attataa 1587 <210> 3 <211> 2100 <212> DNA <213> Unknown <220> <223> GAL10 NT <400> 3 atgacagctc agttacaaag tgaaagtact tctaaaattg ttttggttac aggtggtgct 60 ggatacattg gttcacacac tgtggtagag ctaattgaga atggatatga ctgtgttgtt 120 gctgataacc tgtcgaattc aacttatgat tctgtagcca ggttagaggt cttgaccaag 180 catcacattc ccttctatga ggttgatttg tgtgaccgaa aaggtctgga aaaggttttc 240 aaagaatata aaattgattc ggtaattcac tttgctggtt taaaggctgt aggtgaatct 300 acacaaatcc cgctgagata ctatcacaat aacattttgg gaactgtcgt tttattagag 360 ttaatgcaac aatacaacgt ttccaaattt gttttttcat cttctgctac tgtctatggt 420 gatgctacga gattcccaaa tatgattcct atcccagaag aatgtccctt agggcctact 480 aatccgtatg gtcatacgaa atacgccatt gagaatatct tgaatgatct ttacaatagc 540 gacaaaaaaa gttggaagtt tgctatcttg cgttatttta acccaattgg cgcacatccc 600 tctggattaa tcggagaaga tccgctaggt ataccaaaca atttgttgcc atatatggct 660 caagtagctg ttggtaggcg cgagaagctt tacatcttcg gagacgatta tgattccaga 720 gatggtaccc cgatcaggga ttatatccac gtagttgatc tagcaaaagg tcatattgca 780 gccctgcaat acctagaggc ctacaatgaa aatgaaggtt tgtgtcgtga gtggaacttg 840 ggttccggta aaggttctac agtttttgaa gtttatcatg cattctgcaa agcttctggt 900 attgatcttc catacaaagt tacgggcaga agagcaggtg atgttttgaa cttgacggct 960 aaaccagata gggccaaacg cgaactgaaa tggcagaccg agttgcaggt tgaagactcc 1020 tgcaaggatt tatggaaatg gactactgag aatccttttg gttaccagtt aaggggtgtc 1080 gaggccagat tttccgctga agatatgcgt tatgacgcaa gatttgtgac tattggtgcc 1140 ggcaccagat ttcaagccac gtttgccaat ttgggcgcca gcattgttga cctgaaagtg 1200 aacggacaat cagttgttct tggctatgaa aatgaggaag ggtatttgaa tcctgatagt 1260 gcttatatag gcgccacgat cggcaggtat gctaatcgta tttcgaaggg taagtttagt 1320 ttatgcaaca aagactatca gttaaccgtt aataacggcg ttaatgcgaa tcatagtagt 1380 atcggttctt tccacagaaa aagatttttg ggacccatca ttcaaaatcc ttcaaaggat 1440 gtttttaccg ccgagtacat gctgatagat aatgagaagg acaccgaatt tccaggtgat 1500 ctattggtaa ccatacagta tactgtgaac gttgcccaaa aaagtttgga aatggtatat 1560 aaaggtaaat tgactgctgg tgaagcgacg ccaataaatt taacaaatca tagttatttc 1620 aatctgaaca agccatatgg agacactatt gagggtacgg agattatggt gcgttcaaaa 1680 aaatctgttg atgtcgacaa aaacatgatt cctacgggta atatcgtcga tagagaaatt 1740 gctaccttta actctacaaa gccaacggtc ttaggcccca aaaatcccca gtttgattgt 1800 tgttttgtgg tggatgaaaa tgctaagcca agtcaaatca atactctaaa caatgaattg 1860 acgcttattg tcaaggcttt tcatcccgat tccaatatta cattagaagt tttaagtaca 1920 gagccaactt atcaatttta taccggtgat ttcttgtctg ctggttacga agcaagacaa 1980 ggttttgcaa ttgagcctgg tagatacatt gatgctatca atcaagagaa ctggaaagat 2040 tgtgtaacct tgaaaaacgg tgaaacttac gggtccaaga ttgtctacag attttcctga 2100 2100 <210> 4 <211> 1101 <212> DNA <213> Unknown <220> <223> GAL7 NT <400> 4 atgactgctg aagaatttga tttttctagc cattcccata gacgttacaa tccactaacc 60 gattcatgga tcttagtttc tccacacaga gctaaaagac cttggttagg tcaacaggag 120 gctgcttaca agcccacagc tccattgtat gatccaaaat gctatctatg tcctggtaac 180 aaaagagcta ctggtaacct aaacccaaga tatgaatcaa cgtatatttt ccccaatgat 240 tatgctgccg ttaggctcga tcaacctatt ttaccacaga atgattccaa tgaggataat 300 cttaaaaata ggctgcttaa agtgcaatct gtgagaggca attgtttcgt catatgtttt 360 agccccaatc ataatctaac cattccacaa atgaaacaat cagatctggt tcatattgtt 420 aattcttggc aagcattgac tgacgatctc tccagagaag caagagaaaa tcataagcct 480 ttcaaatatg tccaaatatt tgaaaacaaa ggtacagcca tgggttgttc caacttacat 540 ccacatggcc aagcttggtg cttagaatcc atccctagtg aagtttcgca agaattgaaa 600 tcttttgata aatataaacg tgaacacaat actgatttgt ttgccgatta cgtcaaatta 660 gaatcaagag agaagtcaag agtcgtagtg gagaatgaat cctttattgt tgttgttcca 720 tactgggcca tctggccatt tgagaccttg gtcatttcaa agaagaagct tgcctcaatt 780 agccaattta accaaatggt gaaggaggac ctcgcctcga ttttaaagca actaactatt 840 aagtatgata atttatttga aacgagtttc ccatactcaa tgggtatcca tcaggctcct 900 ttgaatgcga ctggtgatga attgagtaat agttggtttc acatgcattt ctacccacct 960 ttactgagat cagctactgt tcggaaattc ttggttggtt ttgaattgtt aggtgagcct 1020 caaagagatt taacttcgga acaagctgct gaaaaactaa gaaatttaga tggtcagatt 1080 cattatctac aaagactgta a 1101 <210> 5 <211> 1725 <212> DNA <213> Unknown <220> <223> GAL2 NT <400> 5 atggcagttg aggagaacaa tatgcctgtt gtttcacagc aaccccaagc tggtgaagac 60 gtgatctctt cactcagtaa agattcccat ttaagcgcac aatctcaaaa gtattctaat 120 gatgaattga aagccggtga gtcagggtct gaaggctccc aaagtgttcc tatagagata 180 cccaagaagc ccatgtctga atatgttacc gtttccttgc tttgtttgtg tgttgccttc 240 ggcggcttca tgtttggctg ggataccggt actatttctg ggtttgttgt ccaaacagac 300 tttttgagaa ggtttggtat gaaacataag gatggtaccc actatttgtc aaacgtcaga 360 acaggtttaa tcgtcgccat tttcaatatt ggctgtgcct ttggtggtat tatactttcc 420 aaaggtggag atatgtatgg ccgtaaaaag ggtctttcga ttgtcgtctc ggtttatata 480 gttggtatta tcattcaaat tgcctctatc aacaagtggt accaatattt cattggtaga 540 atcatatctg gtttgggtgt cggcggcatc gccgtcttat gtcctatgtt gatctctgaa 600 attgctccaa agcacttgag aggcacacta gtttcttgtt atcagctgat gattactgca 660 ggtatctttt tgggctactg tactaattac ggtacaaaga gctattcgaa ctcagttcaa 720 tggagagttc cattagggct atgtttcgct tggtcattat ttatgattgg cgctttgacg 780 ttagttcctg aatccccacg ttatttatgt gaggtgaata aggtagaaga cgccaagcgt 840 tccattgcta agtctaacaa ggtgtcacca gaggatcctg ccgtccaggc agagttagat 900 ctgatcatgg ccggtataga agctgaaaaa ctggctggca atgcgtcctg gggggaatta 960 ttttccacca agaccaaagt atttcaacgt ttgttgatgg gtgtgtttgt tcaaatgttc 1020 caacaattaa ccggtaacaa ttattttttc tactacggta ccgttatttt caagtcagtt 1080 ggcctggatg attcctttga aacatccatt gtcattggtg tagtcaactt tgcctccact 1140 ttctttagtt tgtggactgt cgaaaacttg ggacatcgta aatgtttact tttgggcgct 1200 gccactatga tggcttgtat ggtcatctac gcctctgttg gtgttactag attatatcct 1260 cacggtaaaa gccagccatc ttctaaaggt gccggtaact gtatgattgt ctttacctgt 1320 ttttatattt tctgttatgc cacaacctgg gcgccagttg cctgggtcat cacagcagaa 1380 tcattcccac tgagagtcaa gtcgaaatgt atggcgttgg cctctgcttc caattgggta 1440 tgggggttct tgattgcatt tttcacccca ttcatcacat ctgccattaa cttctactac 1500 ggttatgtct tcatgggctg tttggttgcc atgttttttt atgtcttttt ctttgttcca 1560 gaaactaaag gcctatcgtt agaagaaatt caagaattat gggaagaagg tgttttacct 1620 tggaaatctg aaggctggat tccttcatcc agaagaggta ataattacga tttagaggat 1680 ttacaacatg acgacaaacc gtggtacaag gccatgctag aataa 1725 <210> 6 <211> 3597 <212> DNA <213> Artificial Sequence <220> <223> GAL1, GAL10, GAL7 deletion sequence <400> 6 tcttggtaat tcctttgcgc tagaattgaa ctcaggtaca atcacttctt ctgaatgaga 60 tttagtcatt atagtttttt ctccttgacg ttaaagtata gaggtatatt aacaattttt 120 tgttgatact tttatgacat ttgaataaga agtaatacaa actgaaaatg ttgaaagtat 180 tagttaaagt ggttatgcag cttttccatt tatatatctg ttaatagatc aaaaatcatc 240 gcttcgctga ttaattaccc cagaaataag gctaaaaaac taatcgcatt atcatcctat 300 ggttgttaat ttgattcgtt aatttgaagg tttgtggggc caggttactg ccaatttttc 360 ctcttcataa ccataaaagc tagtattgta gaatctttat tgttcggagc agtgcggcgc 420 gaggcacatc tgcgtttcag gaacgcgacc ggtgaagacg aggacgcacg gaggagagtc 480 ttccgtcgga gggctgtcgc ccgctcggcg gcttctaatc cgtacttcaa tatagcaatg 540 agcagttaag cgtattactg aaagttccaa agagaaggtt tttttaggct aagataatgg 600 ggctctttac atttccacaa catataagta agattagata tggatatgta tatggtggta 660 atgccatgta atatgattat taaacttctt tgcgtccatc caaaaaaaaa gtaagaattt 720 ttgaaaattc aatataaatg acagctcagt tacaaagtga aagtacttct aaaattgttt 780 tggttacagg tggtgctgga tacattggtt cacacactgt ggtagagcta attgagaatg 840 gatatgactg tgttgttgct gataacctgt cgaattcaac ttatgattct gtagccaggt 900 tagaggtctt gaccaagcat cacattccct tctatgaggt tgatttgtgt gaccgaaaag 960 gtctggaaaa ggttttcaaa gaatataaaa ttgattcggt aattcacttt gctggtttaa 1020 aggctgtagg tgaatctaca caaatcccgc tgagatacta tcacaataac attttgggaa 1080 ctgtcgtttt attagagtta atgcaacaat acaacgtttc caaatttgtt ttttcatctt 1140 ctgctactgt ctatggtgat gctacgagat tcccaaatat gattcctatc ccagaagaat 1200 gtcccttagg gcctactaat ccgtatggtc atacgaaata cgccattgag aatatcttga 1260 atgatcttta caatagcgac aaaaaaagtt ggaagtttgc tatcttgcgt tattttaacc 1320 caattggcgc acatccctct ggattaatcg gagaagatcc gctaggtata ccaaacaatt 1380 tgttgccata tatggctcaa gtagctgttg gtaggcgcga gaagctttac atcttcggag 1440 acgattatga ttccagagat ggtaccccga tcagggatta tatccacgta gttgatctag 1500 caaaaggtca tattgcagcc ctgcaatacc tagaggccta caatgaaaat gaaggtttgt 1560 gtcgtgagtg gaacttgggt tccggtaaag gttctacagt ttttgaagtt tatcatgcat 1620 tctgcaaagc ttctggtatt gatcttccat acaaagttac gggcagaaga gcaggtgatg 1680 ttttgaactt gacggctaaa ccagataggg ccaaacgcga actgaaatgg cagaccgagt 1740 tgcaggttga agactcctgc aaggatttat ggaaatggac tactgagaat ccttttggtt 1800 accagttaag gggtgtcgag gccagatttt ccgctgaaga tatgcgttat gacgcaagat 1860 ttgtgactat tggtgccggc accagatttc aagccacgtt tgccaatttg ggcgccagca 1920 ttgttgacct gaaagtgaac ggacaatcag ttgttcttgg ctatgaaaat gaggaagggt 1980 atttgaatcc tgatagtgct tatataggcg ccacgatcgg caggtatgct aatcgtattt 2040 cgaagggtaa gtttagttta tgcaacaaag actatcagtt aaccgttaat aacggcgtta 2100 atgcgaatca tagtagtatc ggttctttcc acagaaaaag atttttggga cccatcattc 2160 aaaatccttc aaaggatgtt tttaccgccg agtacatgct gatagataat gagaaggaca 2220 ccgaatttcc aggtgatcta ttggtaacca tacagtatac tgtgaacgtt gcccaaaaaa 2280 gtttggaaat ggtatataaa ggtaaattga ctgctggtga agcgacgcca ataaatttaa 2340 caaatcatag ttatttcaat ctgaacaagc catatggaga cactattgag ggtacggaga 2400 ttatggtgcg ttcaaaaaaa tctgttgatg tcgacaaaaa catgattcct acgggtaata 2460 tcgtcgatag agaaattgct acctttaact ctacaaagcc aacggtctta ggccccaaaa 2520 atccccagtt tgattgttgt tttgtggtgg atgaaaatgc taagccaagt caaatcaata 2580 ctctaaacaa tgaattgacg cttattgtca aggcttttca tcccgattcc aatattacat 2640 tagaagtttt aagtacagag ccaacttatc aattttatac cggtgatttc ttgtctgctg 2700 gttacgaagc aagacaaggt tttgcaattg agcctggtag atacattgat gctatcaatc 2760 aagagaactg gaaagattgt gtaaccttga aaaacggtga aacttacggg tccaagattg 2820 tctacagatt ttcctgattt gccagcttac tatccttctt gaaaatatgc actctatatc 2880 ttttagttct taattgcaac acatagattt gctgtataac gaattttatg ctatttttta 2940 aatttggagt tcagtgataa aagtgtcaca gcgaatttcc tcacatgtag ggaccgaatt 3000 gtttacaagt tctctgtacc accatggaga catcaaaaat tgaaaatcta tggaaagata 3060 tggacggtag caacaagaat atagcacgag ccgcggagtt catttcgtta cttttgatat 3120 cactcacaac tattgcgaag cgcttcagtg aaaaaatcat aaggaaaagt tgtaaatatt 3180 attggtagta ttcgtttggt aaagtagagg gggtaatttt tcccctttat tttgttcata 3240 cattcttaaa ttgctttgcc tctccttttg gaaagctata cttcggagca ctgttgagcg 3300 aaggctcatt agatatattt tctgtcattt tccttaaccc aaaaataagg gaaagggtcc 3360 aaaaagcgct cggacaactg ttgaccgtga tccgaaggac tggctataca gtgttcacaa 3420 aatagccaag ctgaaaataa tgtgtagcta tgttcagtta gtttggctag caaagatata 3480 aaagcaggtc ggaaatattt atgggcatta ttatgcagag catcaacatg ataaaaaaaa 3540 acagttgaat attccctcaa aaatgactgc tgaagaattt gatttttcta gccattc 3597 <210> 7 <211> 1781 <212> DNA <213> Artificial Sequence <220> <223> GAL2 deletion sequence <400> 7 cttccggaat ggcttaagta ggttgcaatt tctttttcta ttagtagcta aaaatgggtc 60 acgtgatcta tattcgaaag gggcggttgc ctcaggaagg caccggcggt ctttcgtccg 120 tgcggagata tctgcgccgt tcaggggtcc atgtgccttg gacgatatta aggcagaagg 180 cagtatcggg gcggatcact ccgaaccgag attagttaag cccttcccat ctcaagatgg 240 ggagcaaatg gcattatact cctgctagaa agttaactgt gcacatattc ttaaattata 300 caacattctg gagagctatt gttcaaaaaa caaacatttc gcaggctaaa atgtggagat 360 aggataagtt ttgtagacat atataaacaa tcagtaattg gattgaaaat ttggtgttgt 420 gaattgctct tcattatgca ccttattcaa ttatcatcaa gaatagtaat agttaagtaa 480 acacaagatt aacataataa aaaaaataat tctttcataa tggcagttga ggagaacaat 540 atgcctgttg tttcacagca accccaagct ggtgaagacg tgatctcttc actcagtaaa 600 gattcccatt taagcgcaca atctcaaaag tattctaatg atgaattgaa agccggtgag 660 tcagggtctg aaggctccca aagtgttcct atagagatac ccaagaagcc catgtctgaa 720 tatgttaccg tttccttgct ttgtttgtgt gttgccttcg gcggcttcat gtttggctgg 780 gataccggta ctatttctgg gtttgttgtc caaacagact ttttgagaag gtttggtatg 840 aaacataagg atggtaccca ctatttgtca aacgtcagaa caggtttaat cgtcgccatt 900 ttcaatattg gctgtgcctt tggtggtatt atactttcca aaggtggaga tatgtatggc 960 cgtaaaaagg gtctttcgat tgtcgtctcg gtttatatag ttggtattat cattcaaatt 1020 gcctctatca acaagtggta ccaatatttc attggtagaa tcatatctgg tttgggtgtc 1080 ggcggcatcg ccgtcttatg tcctatgttg atctctgaaa ttgctccaaa gcacttgaga 1140 ggcacactag tttcttgtta tcagctgatg attactgcag gtatcttttt gggctactgt 1200 actaattacg gtacaaagag ctattcgaac tcagttcaat ggagagttcc attagggcta 1260 tgtttcgctt ggtcattatt tatgattggc gctttgacgt tagttcctga atccccacgt 1320 tatttatgtg aggtgaataa ggtagaagac gccaagcgtt ccattgctaa gtctaacaag 1380 gtgtcaccag aggatcctgc cgtccaggca gagttagatc tgatcatggc cggtatagaa 1440 gctgaaaaac tggctggcaa tgcgtcctgg ggggaattat tttccaccaa gaccaaagta 1500 tttcaacgtt tgttgatggg tgtgtttgtt caaatgttcc aacaattaac cggtaacaat 1560 tattttttct actacggtac cgttattttc aagtcagttg gcctggatga ttcctttgaa 1620 acatccattg tcattggtgt agtcaacttt gcctccactt tctttagttt gtggactgtc 1680 gaaaacttgg gacatcgtaa atgtttactt ttgggcgctg ccactatgat ggcttgtatg 1740 gtcatctacg cctctgttgg tgttactaga ttatatcctc a 1781 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAL7 gRNA <400> 8 gattgtaacg tctatgggaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAL2 gRNA <400> 9 caattcggaa agcttccttc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> H978 <400> 10 accctcacag aagacttgca g 21 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> H979 <400> 11 taacgtctat ggccattggc cgaaaagtgc 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> H980 <400> 12 cggccaatgg ccatagacgt tacaatccac 30 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> H981 <400> 13 gaattaacaa tatgaaccag atctg 25 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> H984 <400> 14 tcaactgtta aacgcgacag 20 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> H985 <400> 15 tggcttttac cggaagcttt ccgaattgac 30 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> H986 <400> 16 gaaagcttcc ggtaaaagcc agccatcttc 30 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> H987 <400> 17 cattattcta gcatggcctt g 21 <110> Korea Research Institute of Bioscience and Biotechnology Cellapy Bio Inc. <120> Yeast strain in which all genes involved in galactose utilization are deleted and method for producing recombinant protein using the same <130> KPA210659-KR <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 1308 <212> DNA <213> Unknown <220> <223> GAL80 NT <400> 1 atggactaca acaagagatc ttcggtctca accgtgccta atgcagctcc cataagagtc 60 ggattcgtcg gtctcaacgc agccaaagga tgggcaatca agacacatta ccccgccata 120 ctgcaactat cgtcacaatt tcaaatcact gccttataca gtccaaaaat tgagacttct 180 attgccacca ttcagcgtct aaaattgagt aatgccactg cttttcccac tttagagtca 240 tttgcatcat cttccactat agatatgata gtgatagcta tccaagtggc cagccattat 300 gaagttgtta tgcctctctt ggaattctcc aaaaataatc cgaacctcaa gtatcttttc 360 gtagaatggg cccttgcatg ttcactagat caagccgaat ccatttataa ggctgctgct 420 gaacgtgggg ttcaaaccat catctcttta caaggtcgta aatcaccata tattttgaga 480 gcaaaagaat taatatctca aggctatatc ggcgacatta attcgatcga gattgctgga 540 aatggcggtt ggtacggcta cgaaaggcct gttaaatcac caaaatacat ctatgaaatc 600 gggaacggtg tagatctggt aaccacaaca ttt ggtcaca caatcgatat tttacaatac 660 atgacaagtt cgtacttttc caggataaat gcaatggttt tcaataatat tccagagcaa 720 gagctgatag atgagcgtgg taaccgattg ggccagcgag tcccaaagac agtaccggat 780 catcttttat tccaaggcac attgttaaat ggcaatgttc cagtgtcatg cagtttcaaa 840 ggtggcaaac ctaccaaaaa atttaccaaa aatttggtca ttgacattca cggtaccaag 900 ggagatttga aacttgaagg cgatgccggc ttcgcagaaa tttcaaatct ggtcctttac 960 tacagtggaa ctagagcaaa cgacttcccg ctagccaatg gacaacaagc tcctttagac 1020 ccggggtatg atgcaggtaa agaaatcatg gaagtatatc atttacgaaa ttataatgcc 1080 attgtgggta atattcatcg actgtatcaa tctatctctg acttccactt caatacaaag 1140 aaaattcctg aattaccctc acaatttgta atgcaaggtt tcgatttcga aggctttccc 1200 accttgatgg atgctctgat attacacagg ttaatcgaga gcgtttataa aagtaacatg 1260 atgggctcca cattaaacgt tagcaatatc tcgcattata gtttataa 1308 <210> 2 <211> 1587 <212> DNA <213> Unknown <220> < 223> GAL1 NT <400> 2 atgactaaat ctcattcaga agaagtgatt gtacctgagt tcaattctag cgcaaaggaa 60 ttaccaagac cattggccga aaagtgcccg agcataatta agaaatttat aagcgctt at 120 gatgctaaac cggattttgt tgctagatcg cctggtagag tcaatctaat tggtgaacat 180 attgattatt gtgacttctc ggttttacct ttagctattg attttgatat gctttgcgcc 240 gtcaaagttt tgaacgagaa aaatccatcc attaccttaa taaatgctga tcccaaattt 300 gctcaaagga agttcgattt gccgttggac ggttcttatg tcacaattga tccttctgtg 360 tcggactggt ctaattactt taaatgtggt ctccatgttg ctcactcttt tctaaagaaa 420 cttgcaccgg aaaggtttgc cagtgctcct ctggccgggc tgcaagtctt ctgtgagggt 480 gatgtaccaa ctggcagtgg attgtcttct tcggccgcat tcatttgtgc cgttgcttta 540 gctgttgtta aagcgaatat gggccctggt tatcatatgt ccaagcaaaa tttaatgcgt 600 attacggtcg ttgcagaaca ttatgttggt gttaacaatg gcggtatgga tcaggctgcc 660 tctgtttgcg gtgaggaaga tcatgctcta tacgttgagt tcaaaccgca gttgaaggct 720 actccgttta aatttccgca attaaaaaac catgaaatta gctttgttat tgcgaacacc 780 cttgttgtat ctaacaagtt tgaaaccgcc ccaaccaact ataatttaag agtggtagaa 840 gtcactacag ctgcaaatgt tttagctgcc acgtacggtg ttgttttact ttctggaaaa 900 gaaggatcga gcacgaataa aggtaatcta agagatttca tgaacgttta ttatgccaga 960 tatcacaaca tt tccacacc ctggaacggc gatattgaat ccggcatcga acggttaaca 1020 aagatgctag tactagttga agagtctctc gccaataaga aacagggctt tagtgttgac 1080 gatgtcgcac aatccttgaa ttgttctcgc gaagaattca caagagacta cttaacaaca 1140 tctccagtga gatttcaagt cttaaagcta tatcagaggg ctaagcatgt gtattctgaa 1200 tctttaagag tcttgaaggc tgtgaaatta atgactacag cgagctttac tgccgacgaa 1260 gactttttca agcaatttgg tgccttgatg aacgagtctc aagcttcttg cgataaactt 1320 tacgaatgtt cttgtccaga gattgacaaa atttgttcca ttgctttgtc aaatggatca 1380 tatggttccc gtttgaccgg agctggctgg ggtggttgta ctgttcactt ggttccaggg 1440 ggcccaaatg gcaacataga aaaggtaaaa gaagcccttg ccaatgagtt ctacaaggtc 1500 aagtacccta agatcactga tgctgagcta gaaaatgcta tcatcgtctc taaaccagca 1560 ttgggcagct gtctatatga attataa 1587 <210> 3 <211> 2100 <212> DNA <213> Unknown <220> <223> GAL10 NT <400> 3 atgacagctc agttacaaag tgaaagtact tctaaaattg ttttggttac aggtggtgct 60 ggatacattg gttcacacac tgtggtagag ctaattgaga atggatatga ctgtgttgtt 120 gctgataacc tgtcgaattc aacttatgat tctgtagcca ggttagaggt ct tgaccaag 180 catcacattc ccttctatga ggttgatttg tgtgaccgaa aaggtctgga aaaggttttc 240 aaagaatata aaattgattc ggtaattcac tttgctggtt taaaggctgt aggtgaatct 300 acacaaatcc cgctgagata ctatcacaat aacattttgg gaactgtcgt tttattagag 360 ttaatgcaac aatacaacgt ttccaaattt gttttttcat cttctgctac tgtctatggt 420 gatgctacga gattcccaaa tatgattcct atcccagaag aatgtccctt agggcctact 480 aatccgtatg gtcatacgaa atacgccatt gagaatatct tgaatgatct ttacaatagc 540 gacaaaaaaa gttggaagtt tgctatcttg cgttatttta acccaattgg cgcacatccc 600 tctggattaa tcggagaaga tccgctaggt ataccaaaca atttgttgcc atatatggct 660 caagtagctg ttggtaggcg cgagaagctt tacatcttcg gagacgatta tgattccaga 720 gatggtaccc cgatcaggga ttatatccac gtagttgatc tagcaaaagg tcatattgca 780 gccctgcaat acctagaggc ctacaatgaa aatgaaggtt tgtgtcgtga gtggaacttg 840 ggttccggta aaggttctac agtttttgaa gtttatcatg cattctgcaa agcttctggt 900 attgatcttc catacaaagt tacgggcaga agagcaggtg atgttttgaa cttgacggct 960 aaaccagata gggccaaacg cgaactgaaa tggcagaccg agttgcaggt tgaagactcc 1020 tgcaag gatt tatggaaatg gactactgag aatccttttg gttaccagtt aaggggtgtc 1080 gaggccagat tttccgctga agatatgcgt tatgacgcaa gatttgtgac tattggtgcc 1140 ggcaccagat ttcaagccac gtttgccaat ttgggcgcca gcattgttga cctgaaagtg 1200 aacggacaat cagttgttct tggctatgaa aatgaggaag ggtatttgaa tcctgatagt 1260 gcttatatag gcgccacgat cggcaggtat gctaatcgta tttcgaaggg taagtttagt 1320 ttatgcaaca aagactatca gttaaccgtt aataacggcg ttaatgcgaa tcatagtagt 1380 atcggttctt tccacagaaa aagatttttg ggacccatca ttcaaaatcc ttcaaaggat 1440 gtttttaccg ccgagtacat gctgatagat aatgagaagg acaccgaatt tccaggtgat 1500 ctattggtaa ccatacagta tactgtgaac gttgcccaaa aaagtttgga aatggtatat 1560 aaaggtaaat tgactgctgg tgaagcgacg ccaataaatt taacaaatca tagttatttc 1620 aatctgaaca agccatatgg agacactatt gagggtacgg agattatggt gcgttcaaaa 1680 aaatctgttg atgtcgacaa aaacatgatt cctacgggta atatcgtcga tagagaaatt 1740 gctaccttta actctacaaa gccaacggtc ttaggcccca aaaatcccca gtttgattgt 1800 tgttttgtgg tggatgaaaa tgctaagcca agtcaaatca atactctaaa caatgaattg 1860 acgcttattg t caaggcttt tcatcccgat tccaatatta cattagaagt tttaagtaca 1920 gagccaactt atcaatttta taccggtgat ttcttgtctg ctggttacga agcaagacaa 1980 ggttttgcaa ttgagcctgg tagatacatt gatgctatca atcaagagaa ctggaaagat 2040 tgtgtaacct tgaaaaacgg tgaaacttac gggtccaaga ttgtctacag attttcctga 2100 2100 <210> 4 <211> 1101 <212> DNA <213> Unknown <220> <223> GAL7 NT <400> 4 atgactgctg aagaatttga tttttctagc cattcccata gacgttacaa tccactaacc 60 gattcatgga tcttagtttc tccacacaga gctaaaagac cttggttagg tcaacaggag 120 gctgcttaca agcccacagc tccattgtat gatccaaaat gctatctatg tcctggtaac 180 aaaagagcta ctggtaacct aaacccaaga tatgaatcaa cgtatatttt ccccaatgat 240 tatgctgccg ttaggctcga tcaacctatt ttaccacaga atgattccaa tgaggataat 300 cttaaaaata ggctgcttaa agtgcaatct gtgagaggca attgtttcgt catatgtttt 360 agccccaatc ataatctaac cattccacaa atgaaacaat cagatctggt tcatattgtt 420 aattcttggc aagcattgac tgacgatctc tccagagaag caagagaaaa tcataagcct 480 ttcaaatatg tccaaatatt tgaaaacaaa ggtacagcca tgggttgttc caacttacat 540 ccacatggtg cagcttggtcc ttagaatcc atccctagtg aagtttcgca agaattgaaa 600 tcttttgata aatataaacg tgaacacaat actgatttgt ttgccgatta cgtcaaatta 660 gaatcaagag agaagtcaag agtcgtagtg gagaatgaat cctttattgt tgttgttcca 720 tactgggcca tctggccatt tgagaccttg gtcatttcaa agaagaagct tgcctcaatt 780 agccaattta accaaatggt gaaggaggac ctcgcctcga ttttaaagca actaactatt 840 aagtatgata atttatttga aacgagtttc ccatactcaa tgggtatcca tcaggctcct 900 ttgaatgcga ctggtgatga attgagtaat agttggtttc acatgcattt ctacccacct 960 ttactgagat cagctactgt tcggaaattc ttggttggtt ttgaattgtt aggtgagcct 1020 caaagagatt taacttcgga acaagctgct gaaaaactaa gaaatttaga tggtcagatt 1080 cattatctac aaagactgta a 1101 <210> 5 <211> 1725 <212> DNA <213> Unknown <220> <223> GAL2 NT <400> 5 atggcagttg aggagaacaa tatgcctgtt gtttcacagc aaccccaagc tggtgaagac 60 gtgatctctt cactcagtaa agattcccat ttaagcgcac aatctcaaaa gtattctaat 120 gatgaattga aagccggtga gtcagggtct gaaggctccc aaagtgttcc tatagagata 180 cccaagaagc ccatgtctga atatgttacc gtttccttgc tttgtttgtg tgttgccttggc 2 ct4tccttggc gtttggctg ggataccggt actatttctg ggtttgttgt ccaaacagac 300 tttttgagaa ggtttggtat gaaacataag gatggtaccc actatttgtc aaacgtcaga 360 acaggtttaa tcgtcgccat tttcaatatt ggctgtgcct ttggtggtat tatactttcc 420 aaaggtggag atatgtatgg ccgtaaaaag ggtctttcga ttgtcgtctc ggtttatata 480 gttggtatta tcattcaaat tgcctctatc aacaagtggt accaatattt cattggtaga 540 atcatatctg gtttgggtgt cggcggcatc gccgtcttat gtcctatgtt gatctctgaa 600 attgctccaa agcacttgag aggcacacta gtttcttgtt atcagctgat gattactgca 660 ggtatctttt tgggctactg tactaattac ggtacaaaga gctattcgaa ctcagttcaa 720 tggagagttc cattagggct atgtttcgct tggtcattat ttatgattgg cgctttgacg 780 ttagttcctg aatccccacg ttatttatgt gaggtgaata aggtagaaga cgccaagcgt 840 tccattgcta agtctaacaa ggtgtcacca gaggatcctg ccgtccaggc agagttagat 900 ctgatcatgg ccggtataga agctgaaaaa ctggctggca atgcgtcctg gggggaatta 960 ttttccacca agaccaaagt atttcaacgt ttgttgatgg gtgtgtttgt tcaaatgttc 1020 caacaattaa ccggtaacaa ttattttttc tactacggta ccgttatttt caagtcagtt 1080 ggcctggatg attcctttga aacatcca tt gtcattggtg tagtcaactt tgcctccact 1140 ttctttagtt tgtggactgt cgaaaacttg ggacatcgta aatgtttact tttgggcgct 1200 gccactatga tggcttgtat ggtcatctac gcctctgttg gtgttactag attatatcct 1260 cacggtaaaa gccagccatc ttctaaaggt gccggtaact gtatgattgt ctttacctgt 1320 ttttatattt tctgttatgc cacaacctgg gcgccagttg cctgggtcat cacagcagaa 1380 tcattcccac tgagagtcaa gtcgaaatgt atggcgttgg cctctgcttc caattgggta 1440 tgggggttct tgattgcatt tttcacccca ttcatcacat ctgccattaa cttctactac 1500 ggttatgtct tcatgggctg tttggttgcc atgttttttt atgtcttttt ctttgttcca 1560 gaaactaaag gcctatcgtt agaagaaatt caagaattat gggaagaagg tgttttacct 1620 tggaaatctg aaggctggat tccttcatcc agaagaggta ataattacga tttagaggat 1680 ttacaacatg acgacaaacc gtggtacaag gccatgctag aataa 1725 <210> 6 <211> 3597 <212> DNA <213> Artificial Sequence <220> <223> GAL1, GAL10 , GAL7 deletion sequence <400> 6 tcttggtaat tcctttgcgc tagaattgaa ctcaggtaca atcacttctt ctgaatgaga 60 tttagtcatt atagtttttt ctccttgacg ttaaagtata gaggtatatt aacaattttt 120 tgttgatact ttta tgacat ttgaataaga agtaatacaa actgaaaatg ttgaaagtat 180 tagttaaagt ggttatgcag cttttccatt tatatatctg ttaatagatc aaaaatcatc 240 gcttcgctga ttaattaccc cagaaataag gctaaaaaac taatcgcatt atcatcctat 300 ggttgttaat ttgattcgtt aatttgaagg tttgtggggc caggttactg ccaatttttc 360 ctcttcataa ccataaaagc tagtattgta gaatctttat tgttcggagc agtgcggcgc 420 gaggcacatc tgcgtttcag gaacgcgacc ggtgaagacg aggacgcacg gaggagagtc 480 ttccgtcgga gggctgtcgc ccgctcggcg gcttctaatc cgtacttcaa tatagcaatg 540 agcagttaag cgtattactg aaagttccaa agagaaggtt tttttaggct aagataatgg 600 ggctctttac atttccacaa catataagta agattagata tggatatgta tatggtggta 660 atgccatgta atatgattat taaacttctt tgcgtccatc caaaaaaaaa gtaagaattt 720 ttgaaaattc aatataaatg acagctcagt tacaaagtga aagtacttct aaaattgttt 780 tggttacagg tggtgctgga tacattggtt cacacactgt ggtagagcta attgagaatg 840 gatatgactg tgttgttgct gataacctgt cgaattcaac ttatgattct gtagccaggt 900 tagaggtctt gaccaagcat cacattccct tctatgaggt tgatttgtgt gaccgaaaag 960 gtctggaaaa ggttttcaaa gaatataaaa tt gattcggt aattcacttt gctggtttaa 1020 aggctgtagg tgaatctaca caaatcccgc tgagatacta tcacaataac attttgggaa 1080 ctgtcgtttt attagagtta atgcaacaat acaacgtttc caaatttgtt ttttcatctt 1140 ctgctactgt ctatggtgat gctacgagat tcccaaatat gattcctatc ccagaagaat 1200 gtcccttagg gcctactaat ccgtatggtc atacgaaata cgccattgag aatatcttga 1260 atgatcttta caatagcgac aaaaaaagtt ggaagtttgc tatcttgcgt tattttaacc 1320 caattggcgc acatccctct ggattaatcg gagaagatcc gctaggtata ccaaacaatt 1380 tgttgccata tatggctcaa gtagctgttg gtaggcgcga gaagctttac atcttcggag 1440 acgattatga ttccagagat ggtaccccga tcagggatta tatccacgta gttgatctag 1500 caaaaggtca tattgcagcc ctgcaatacc tagaggccta caatgaaaat gaaggtttgt 1560 gtcgtgagtg gaacttgggt tccggtaaag gttctacagt ttttgaagtt tatcatgcat 1620 tctgcaaagc ttctggtatt gatcttccat acaaagttac gggcagaaga gcaggtgatg 1680 ttttgaactt gacggctaaa ccagataggg ccaaacgcga actgaaatgg cagaccgagt 1740 tgcaggttga agactcctgc aaggatttat ggaaatggac tactgagaat ccttttggtt 1800 accagttaag gggtgtcgag gccagatttt ccgctgaa ga tatgcgttat gacgcaagat 1860 ttgtgactat tggtgccggc accagatttc aagccacgtt tgccaatttg ggcgccagca 1920 ttgttgacct gaaagtgaac ggacaatcag ttgttcttgg ctatgaaaat gaggaagggt 1980 atttgaatcc tgatagtgct tatataggcg ccacgatcgg caggtatgct aatcgtattt 2040 cgaagggtaa gtttagttta tgcaacaaag actatcagtt aaccgttaat aacggcgtta 2100 atgcgaatca tagtagtatc ggttctttcc acagaaaaag atttttggga cccatcattc 2160 aaaatccttc aaaggatgtt tttaccgccg agtacatgct gatagataat gagaaggaca 2220 ccgaatttcc aggtgatcta ttggtaacca tacagtatac tgtgaacgtt gcccaaaaaa 2280 gtttggaaat ggtatataaa ggtaaattga ctgctggtga agcgacgcca ataaatttaa 2340 caaatcatag ttatttcaat ctgaacaagc catatggaga cactattgag ggtacggaga 2400 ttatggtgcg ttcaaaaaaa tctgttgatg tcgacaaaaa catgattcct acgggtaata 2460 tcgtcgatag agaaattgct acctttaact ctacaaagcc aacggtctta ggccccaaaa 2520 atccccagtt tgattgttgt tttgtggtgg atgaaaatgc taagccaagt caaatcaata 2580 ctctaaacaa tgaattgacg cttattgtca aggcttttca tcccgattcc aatattacat 2640 tagaagtttt aagtacagag ccaacttatc aattttatac cgg tgatttc ttgtctgctg 2700 gttacgaagc aagacaaggt tttgcaattg agcctggtag atacattgat gctatcaatc 2760 aagagaactg gaaagattgt gtaaccttga aaaacggtga aacttacggg tccaagattg 2820 tctacagatt ttcctgattt gccagcttac tatccttctt gaaaatatgc actctatatc 2880 ttttagttct taattgcaac acatagattt gctgtataac gaattttatg ctatttttta 2940 aatttggagt tcagtgataa aagtgtcaca gcgaatttcc tcacatgtag ggaccgaatt 3000 gtttacaagt tctctgtacc accatggaga catcaaaaat tgaaaatcta tggaaagata 3060 tggacggtag caacaagaat atagcacgag ccgcggagtt catttcgtta cttttgatat 3120 cactcacaac tattgcgaag cgcttcagtg aaaaaatcat aaggaaaagt tgtaaatatt 3180 attggtagta ttcgtttggt aaagtagagg gggtaatttt tcccctttat tttgttcata 3240 cattcttaaa ttgctttgcc tctccttttg gaaagctata cttcggagca ctgttgagcg 3300 aaggctcatt agatatattt tctgtcattt tccttaaccc aaaaataagg gaaagggtcc 3360 aaaaagcgct cggacaactg ttgaccgtga tccgaaggac tggctataca gtgttcacaa 3420 aatagccaag ctgaaaataa tgtgtagcta tgttcagtta gtttggctag caaagatata 3480 aaagcaggtc ggaaatattt atgggcatta ttatgcagag catcaacat g ataaaaaaaa 3540 acagttgaat attccctcaa aaatgactgc tgaagaattt gatttttcta gccattc 3597 <210> 7 <211> 1781 <212> DNA <213> Artificial Sequence <220> <223> GAL2 deletion sequence <400> 7 cttccggaat ggcttaagta ggttgcaatt tctttttcta ttagtagcta aaaatgggtc 60 acgtgatcta tattcgaaag gggcggttgc ctcaggaagg caccggcggt ctttcgtccg 120 tgcggagata tctgcgccgt tcaggggtcc atgtgccttg gacgatatta aggcagaagg 180 cagtatcggg gcggatcact ccgaaccgag attagttaag cccttcccat ctcaagatgg 240 ggagcaaatg gcattatact cctgctagaa agttaactgt gcacatattc ttaaattata 300 caacattctg gagagctatt gttcaaaaaa caaacatttc gcaggctaaa atgtggagat 360 aggataagtt ttgtagacat atataaacaa tcagtaattg gattgaaaat ttggtgttgt 420 gaattgctct tcattatgca ccttattcaa ttatcatcaa gaatagtaat agttaagtaa 480 acacaagatt aacataataa aaaaaataat tctttcataa tggcagttga ggagaacaat 540 atgcctgttg tttcacagca accccaagct ggtgaagacg tgatctcttc actcagtaaa 600 gattcccatt taagcgcaca atctcaaaag tattctaatg atgaattgaa agccggtgag 660 tcagggtctg aaggctccca aagtgttcct atagagatac cca agaagcc catgtctgaa 720 tatgttaccg tttccttgct ttgtttgtgt gttgccttcg gcggcttcat gtttggctgg 780 gataccggta ctatttctgg gtttgttgtc caaacagact ttttgagaag gtttggtatg 840 aaacataagg atggtaccca ctatttgtca aacgtcagaa caggtttaat cgtcgccatt 900 ttcaatattg gctgtgcctt tggtggtatt atactttcca aaggtggaga tatgtatggc 960 cgtaaaaagg gtctttcgat tgtcgtctcg gtttatatag ttggtattat cattcaaatt 1020 gcctctatca acaagtggta ccaatatttc attggtagaa tcatatctgg tttgggtgtc 1080 ggcggcatcg ccgtcttatg tcctatgttg atctctgaaa ttgctccaaa gcacttgaga 1140 ggcacactag tttcttgtta tcagctgatg attactgcag gtatcttttt gggctactgt 1200 actaattacg gtacaaagag ctattcgaac tcagttcaat ggagagttcc attagggcta 1260 tgtttcgctt ggtcattatt tatgattggc gctttgacgt tagttcctga atccccacgt 1320 tatttatgtg aggtgaataa ggtagaagac gccaagcgtt ccattgctaa gtctaacaag 1380 gtgtcaccag aggatcctgc cgtccaggca gagttagatc tgatcatggc cggtatagaa 1440 gctgaaaaac tggctggcaa tgcgtcctgg ggggaattat tttccaccaa gaccaaagta 1500 tttcaacgtt tgttgatggg tgtgtttgtt caaatgttcc aacaattaac cgg taacaat 1560 tattttttct actacggtac cgttattttc aagtcagttg gcctggatga ttcctttgaa 1620 acatccattg tcattggtgt agtcaacttt gcctccactt tctttagttt gtggactgtc 1680 gaaaacttgg gacatcgtaa atgtttactt ttgggcgctg ccactatgat ggcttgtatg 1740 gtcatctacg cctctgttgg tgttactaga ttatatcctc a 1781 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAL7 gRNA <400> 8 gattgtaacg tctatgggaa 20 <210> 9 < 211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAL2 gRNA <400> 9 caattcggaa agcttccttc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> H978 <400> 10 accctcacag aagacttgca g 21 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> H979 <400> 11 taacgtctat ggccattggc cgaaaagtgc 30 <210> 12 <211> 30 < 212> DNA <213> Artificial Sequence <220> <223> H980 <400> 12 cggccaatgg ccatagacgt tacaatccac 30 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> H981 <400> 13 gaattaacaa tatgaaccag atctg 25 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> H984 <400> 14 tcaactgtta aacgcgacag 20 <210> 15 <211> 30 <212> DNA <213 > Artificial Sequence <220> <223> H985 <400> 15 tggcttttac cggaagcttt ccgaa ttgac 30 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> H986 <400> 16 gaaagcttcc ggtaaaagcc agccatcttc 30 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> H987<400> 17 cattattcta gcatggcctt g 21
Claims (7)
Yeast mutant strain for expression of recombinant protein, in which the GAL80 gene and the gene related to galactose metabolism are deleted.
The mutant yeast according to claim 1, wherein the galactose metabolism related gene is any one or more selected from the group consisting of GAL1, GAL10, GAL7 and GAL2 genes.
The yeast mutant according to claim 1, wherein the yeast has galactose-fermenting ability or lactose-fermenting ability.
According to claim 1, wherein the yeast saccharomyces genus ( Saccharomyces sp.) And Cluj Vero My Seth ( Kluyveromyce sp.) Will any one or more selected from the group consisting of, yeast mutant strain.
b) 상기 a)의 형질전환된 효모 변이주를 갈락토스 결핍 배지에서 배양하고 재조합 단백질을 발현시키는 단계;를 포함하는, 재조합 단백질 생산 방법.
a) transforming the yeast mutant strain of claim 1 with a recombinant protein expression vector containing a gene encoding a foreign protein of interest; and
b) culturing the transformed yeast mutant of a) in a galactose-deficient medium and expressing a recombinant protein;
The method of claim 5, wherein the vector of step a) includes a promoter of one or more genes selected from the group consisting of GAL1, GAL10, GAL7 and GAL2.
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| KR20090016364A (en) * | 2007-08-10 | 2009-02-13 | 한국생명공학연구원 | A method for producing a recombinant protein using a gal80 deficient yeast strain |
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| KR20090016364A (en) * | 2007-08-10 | 2009-02-13 | 한국생명공학연구원 | A method for producing a recombinant protein using a gal80 deficient yeast strain |
Non-Patent Citations (2)
| Title |
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| APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2005, Vol. 71, No. 11, pp. 6465-6472.* * |
| SCIENCE, 2001, Vol. 292, pp. 929-934.* * |
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