KR100426286B1 - Process for the preparation of human interleukin-11 in yeast - Google Patents
Process for the preparation of human interleukin-11 in yeast Download PDFInfo
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Abstract
Description
본 발명은 효모를 이용한 인간 인터루킨(interleukin)-11 단백질의 제조방법에 관한 것으로, 더욱 상세하게는 인간 인터루킨-11의 염기서열을 효모에서 선호하는 염기코돈으로 아미노산 변화없이 치환하고 유비퀴틴 유전자와 융합하여 발현시킴으로써 인간 인터루킨-11 단백질을 높은 효율로 생산하는 방법에 관한 것이다.The present invention relates to a method for producing human interleukin-11 protein using yeast, and more particularly, to replace the nucleotide sequence of human interleukin-11 with a preferred base codon in yeast without changing amino acids and fusion with ubiquitin gene. By expressing it relates to a method for producing human interleukin-11 protein with high efficiency.
인터루킨-11은 영장류 골수 기질세포인 PU-34의 배양액에서 처음 발견되었고 간엽유래 부착성 세포에서 생성되는 다기능 싸이토카인(cytokins)이다. COS-1 세포에서 발현되는 인터루킨-11은 Ig-G 분비 B 세포의 생성을 자극하고(Musashi, M. et al., PNAS U.S.A. 88, 765(1991)), 조혈성 모체(Dexter T. M. et al., J. Cell Physical., 83, 461(1973))와 거핵세포(megakaryotes)를 증가시키는데, 이 때 인터루킨-3과 함께 처리하면 상승작용을 일으킨다고 알려져 있다(Bruno. E. et al., Exp. Hematol., 19, 378(1991)). 또한 인터루킨-11은 지질생성(adipogenesis) 억제제로써 지질세포 분화 및 리포단백 리파제 활성을 억제하고, 부분적으로 활성이 있는 적골수(red marrow)를 불활성의 황골수(yellow marrow)로 전환시키는 작용을 중재함으로써 골수의 혈액병리 활성을 조절한다(Ohsumi. et al., FEBS letter. 288, 13-16(1991)).Interleukin-11 is a multifunctional cytokine first discovered in culture of primate bone marrow stromal cells, PU-34, and produced in mesenchymal-derived adherent cells. Interleukin-11 expressed in COS-1 cells stimulates the production of Ig-G secreted B cells (Musashi, M. et al., PNAS USA 88, 765 (1991)), and hematopoietic matrix (Dexter ™ et al. , J. Cell Physical., 83, 461 (1973)) and megakaryotes, which are known to be synergistic when treated with interleukin-3 (Bruno. E. et al., Exp Hematol., 19, 378 (1991). Interleukin-11 also inhibits lipid cell differentiation and lipoprotein lipase activity as an adipogenesis inhibitor and mediates the action of converting partially active red marrow into inactive yellow marrow. Thereby regulating blood pathological activity of bone marrow (Ohsumi. Et al., FEBS letter. 288, 13-16 (1991)).
또한 인터루킨-11은 해마 모체(hippocampal progenitor)의 분화에도 관여하는 것으로 알려졌다(Mehler. et al., Nature, 362, 62(1993)). 이의 cDNA 서열은 폴의 골수 기질세포주 PU-34로부터 밝혀졌고(Paul. et al., Proc. Natl, Acad. Sci. U.S.A. 27, 7512(1990)), 인터루킨-11과 동일한 것으로 밝혀진 지질생성 억제인자(AGIF)도 알려져 있다(Kawashima, I. et al., FEBS Letter 283, 199-202(1991)).Interleukin-11 is also known to be involved in the differentiation of the hippocampal progenitor (Mehler. Et al., Nature, 362, 62 (1993)). Its cDNA sequence was identified from Paul's bone marrow stromal cell line PU-34 (Paul. Et al., Proc. Natl, Acad. Sci. USA 27, 7512 (1990)) and was found to be the same as the lipidogenesis inhibitor that was found to be identical to interleukin-11. (AGIF) is also known (Kawashima, I. et al., FEBS Letter 283, 199-202 (1991)).
성숙한 인터루킨-11은 178개의 아미노산에 당화부위가 없는 단백질로써 COS-1 세포에서 발현시 약 20 kDa(Paul. S. R. et al., Proc. Natl. Acad. Sci. U.S.A. 87, 7512(1990)), 또는 23 kDa(Ohsumi. J. et al., FEBS Letter 288, 13-16(1991)) 크기의 단백질로 생성된다고 보고되었다. 한편, 대장균에서 생성된 rhu IL-11는2.5 x 106U/mg 단백질의 활성을 나타낸다(Hangol G. et al., Blood. 81, 965-972(1993)).Mature interleukin-11 is a protein with no glycosylation site at 178 amino acids and is expressed in COS-1 cells at about 20 kDa (Paul. SR et al., Proc. Natl. Acad. Sci. USA 87, 7512 (1990)), Or 23 kDa (Ohsumi. J. et al., FEBS Letter 288, 13-16 (1991)). On the other hand, rhu IL-11 produced in Escherichia coli shows an activity of 2.5 × 10 6 U / mg protein (Hangol G. et al., Blood. 81, 965-972 (1993)).
본 발명자들은 섬유아세포주(fibroblast cell line) MRC-5(ATCC CCL 171) 세포로부터 mRNA를 추출하여 cDNA를 합성하고 이를 그대로 대장균과 효모세포에 클로닝한 결과 그 발현 정도가 지극히 낮다는 것을 발견하여 이를 개선하기 위한 연구를 계속하던 중, 인터루킨-11 유전자의 염기서열(Sharp, R. M. et al., Natl. Acad. Res. 14, 5125-5143(1986))을 효모 선호코돈으로 아미노산의 변화없이 치환변형시키고, 유비퀴틴(Ecker, D. J. et al., J. Biol. Chem. 264, 7715-7719(1989)) 유전자 다음에 융합시켜 발현시킬 때 인간 인터루킨-11 단백질을 높은 발현율로 생산할 수 있음을 발견하여 본 발명의 완성에 이르게 되었다.The inventors found that mRNA was extracted from fibroblast cell line MRC-5 (ATCC CCL 171) cells to synthesize cDNA and cloned into E. coli and yeast cells. While continuing research to improve the nucleotide sequence of the interleukin-11 gene (Sharp, RM et al., Natl. Acad. Res. 14, 5125-5143 (1986)), the yeast preference codon was used as a modification without substitution of amino acids. And the expression of the ubiquitin (Ecker, DJ et al., J. Biol. Chem. 264, 7715-7719 (1989)) gene followed by fusion to produce a high expression rate of human interleukin-11 protein. The invention has been completed.
본 발명의 목적은 효모세포를 이용한 천연형 인간 인터루킨-11 단백질의 생산방법을 제공하는 것이다.It is an object of the present invention to provide a method for producing natural human interleukin-11 protein using yeast cells.
본 발명의 다른 목적은 효모세포로부터 천연형 인간 인터루킨-11 단백질을 생산하는데 사용되는 인간 인터루킨-11의 변형된 재조합 cDNA, 이를 포함하는 발현벡터 및 상기 발현벡터로 형질전환된 형질전환체를 제공하는 것이다.It is another object of the present invention to provide a modified recombinant cDNA of human interleukin-11, an expression vector comprising the same, and a transformant transformed with the expression vector, which are used to produce a native human interleukin-11 protein from yeast cells. will be.
상기 목적에 따라, 본 발명에서는 하기 염기서열을 갖는 인간 인터루킨-11를코드하는 재조합 cDNA:In accordance with the above object, in the present invention, recombinant cDNA encoding human interleukin-11 having the following nucleotide sequence:
상기 cDNA를 포함하는 효모용 인간 인터루킨-11 발현벡터 및 상기 발현벡터로 형질전환된 효모세포주를 제공한다.It provides a human interleukin-11 expression vector for yeast comprising the cDNA and a yeast cell line transformed with the expression vector.
또한 본 발명에서는 상기 효모세포를 배양하여 천연형 인간 인터루킨-11을 회수하는 것을 포함하는 인간 인터루킨-11 단백질의 제조방법을 제공한다.In another aspect, the present invention provides a method for producing human interleukin-11 protein comprising culturing the yeast cells to recover native human interleukin-11.
본 발명에서는 효모세포 내에서 인간 인터루킨-11 단백질을 발현시키는데 최적화하기 위해 하기와 같은 인터루킨-11 유전자의 cDNA 염기서열에서 표시된 위치를 효모 선호코돈으로 아미노산 서열에는 변화가 없도록 치환함으로써 인간 인터루킨 단백질의 발현율을 증가시킬 수 있었다.In the present invention, in order to optimize the expression of human interleukin-11 protein in yeast cells, the expression rate of the human interleukin protein is replaced by replacing the amino acid sequence with the yeast preference codon so that the position indicated in the cDNA sequence of the interleukin-11 gene is not changed. Could increase.
본 발명의 변형된 인간 인터루킨-11 유전자를 효모세포 내에서 유비퀴틴 융합단백질로 발현되도록 한 후 이것이 효모세포 내에서 절단되어 천연형 인간 인터루킨-11 단백질로 생산되도록 할 수 있다. 즉, 변형된 인간 인터루킨-11 유전자를 효모의 유비퀴틴 유전자 다음에 융합시키면, 이로 인해 형질전환된 효모세포 내에서 유비퀴틴 절단효소에 의해 유비퀴틴 단백질과 절단된 인간 인터루킨-11 단백질로 발현될 수 있다. 이 때 인터루킨-11 처럼 아미노 말단의 아미노산이프롤린(proline)인 경우 유비퀴틴 절단효소에 의해 절단이 되지 않기 때문에 인터루킨-11 유전자의 전구서열의 아미노 말단에 알라닌 코돈을 첨가한 다음 위와 같이 유비퀴틴 유전자와 융합시킨다. 이와 같이 제조된 발현벡터로 형질전환된 효모세포에서는 단일한 크기의 인간 인터루킨-11 단백질을 생산할 수 있음을 확인하였다.The modified human interleukin-11 gene of the present invention can be expressed as a ubiquitin fusion protein in yeast cells, which can then be cleaved in yeast cells to produce a native human interleukin-11 protein. That is, when the modified human interleukin-11 gene is fused after the ubiquitin gene of yeast, it can be expressed in the transformed yeast cells as ubiquitin protein and cleaved human interleukin-11 protein by ubiquitin cleavage enzyme. In this case, since the amino acid proline is not cleaved by ubiquitin cleavage enzyme, as in interleukin-11, alanine codon is added to the amino terminal of the precursor sequence of interleukin-11 gene and then fused with ubiquitin gene as above. Let's do it. It was confirmed that the yeast cells transformed with the expression vector thus prepared could produce human interleukin-11 protein of a single size.
이하, 본 발명을 하기 실시예에 의거하여 좀더 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.
비교예 : cDNA 유래 인간 IL-11 유전자의 발현Comparative Example: Expression of Human IL-11 Gene Derived from cDNA
(단계 1) 인간 MRC-5 세포로부터 인간 IL-11 유전자의 분리(Step 1) Isolation of Human IL-11 Gene from Human MRC-5 Cells
인간의 MRC-5 세포(ATCC CCL 171, Lung diploid)를 250U(0.5㎍/㎖)의 인터루킨-1α로 24 시간 이상 처리한 후, 콜로진스키등의 방법(Cholozynski, P., et al., Anal. Biochem. 162, 156(1987))에 따라 세포 침전물로부터 mRNA 추출을 다음과 같이 수행하였다. 5 x 106세포에 RNA 추출액(4M 구아니디움 티오시아네이트, 25mM 구연산나트륨, pH 7.0, 0.5% 사르코실, 0.1M 2-머캅토에탄올) 1㎖를 가하고 진탕시켜 세포막을 파괴시킨 다음, 2M 아세트산 나트륨(pH 4.0) 0.8㎖, 페놀 0.8㎖, 클로로포름-이소아밀알콜(49:1 v/v) 1.6㎖를 처리한 후 원심분리하였다. 상층의 수용액에 이소프로판올을 처리하여 RNA 침전물을 얻어 20㎕의 TE 완충액(10mM Tris-HCl, pH 7.5, 1mM EDTA)에 용존시켰다.Human MRC-5 cells (ATCC CCL 171, Lung diploid) were treated with 250U (0.5 μg / ml) of interleukin-1α for at least 24 hours, followed by the method of Korozinski et al. (Cholozynski, P., et al., Anal. Biochem. 162, 156 (1987)), mRNA extraction from cell precipitate was performed as follows. 1 ml of RNA extract (4M guanidium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1M 2-mercaptoethanol) was added to 5 x 10 6 cells and shaken to break the cell membrane, followed by 2M 0.8 ml of sodium acetate (pH 4.0), 0.8 ml of phenol, and 1.6 ml of chloroform-isoamyl alcohol (49: 1 v / v) were treated and then centrifuged. The aqueous solution of the upper layer was treated with isopropanol to obtain an RNA precipitate, which was dissolved in 20 µl of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).
mRNA로부터 cDNA 단일가닥을 합성하기 위해서, 상기에서 얻은 RNA 용액 10㎕를 취하여 0.1M 메틸머큐리(CH3HgOH) 2㎕를 가한 다음 상온에서 10 분간 방치하여 RNA의 2차 구조를 풀어준 후 1M의 2-머캅토에탄올 2㎕를 가하여 5 분간 반응시켰다. 여기에 10배 농축 역전사 효소 반응용액(500mM Tris-HCl, pH 8.3, 750mM KCl, 30mM MgCl2, 10mM Dithiothreitol) 5㎕, 10mM dNTP 혼합액(dGTP, dATP, dTTP 및 dCTP가 각각 10mM) 2.5㎕, 디에틸피로카보네이트(DEPC, diethylpyrocarbonate) 24㎕가 처리된 증류수, RNase 억제제(RNasin, 1U/㎕, Promega, U.S.A.) 1.0㎕, 올리고(dT)12-18 시발체(oligo(dT)12-18 primer, GibcoBRL, U.S.A.) 1㎕를 가하고 상온에서 10 분간 방치시켜 RNA 주형과 시발체가 잘 붙게 한 다음, 2.5㎕의 역전사 효소(18U/㎕, Superscript RNase H- Reverse transcriptase, BRL, U.S.A.)를 가하여 37℃에서 1 시간 동안 반응시켜 cDNA 가닥을 합성하였다.To synthesize cDNA single strands from mRNA, 10 μl of the RNA solution obtained above was taken, 2 μl of 0.1M methyl mercury (CH 3 HgOH) was added, and left at room temperature for 10 minutes to release the secondary structure of RNA. 2 µl of 2-mercaptoethanol was added and allowed to react for 5 minutes. Here, 5 μl of a 10-fold concentrated reverse transcriptase reaction solution (500 mM Tris-HCl, pH 8.3, 750 mM KCl, 30 mM MgCl 2 , 10 mM Dithiothreitol), 2.5 μl of 10 mM dNTP mixed solution (10 mM of dGTP, dATP, dTTP and dCTP, respectively) Distilled water treated with 24 μl of ethylpyrocarbonate (DEPC), 1.0 μl of RNase inhibitor (RNasin, 1U / μl, Promega, USA), oligo (dT) 12-18 primer, GibcoBRL , USA) 1 μl and allowed to stand at room temperature for 10 minutes to allow RNA template and primer to adhere well, and then 2.5 μl reverse transcriptase (18 U / μl, Superscript RNase H- Reverse transcriptase, BRL, USA) was added at 37 ° C. The reaction was performed for a time to synthesize cDNA strands.
상기 cDNA로부터 인간 인터루킨-11 유전자를 중합효소 연쇄반응을 이용해 증폭시키기 위해 두개의 올리고뉴클레오티드 프라어머를 핵산합성기(DNA Synthesizer, Applied Biosystem Inc., U.S.A.)를 이용하여 합성하였다.Two oligonucleotide primers were synthesized using a nucleic acid synthesizer (DNA Synthesizer, Applied Biosystem Inc., U.S.A.) to amplify the human interleukin-11 gene from the cDNA using a polymerase chain reaction.
부터 메티오닌을 포함하는 인간 인터루킨-11 유전자와 동일한 염기서열Sequences identical to those of the human interleukin-11 gene including methionine
개의 종료코돈을 갖고 있으며 17번째 헥산부터는 인간 인터루킨-11의 카복시 말단염기와 상보적인 서열을 갖도록 고안하였다.It has a dog codon and is designed to have a sequence complementary to the carboxy terminal base of human interleukin-11 from 17th hexane.
cDNA 가닥을 주형으로 하여 중합효소 연쇄반응을 수행하되, 상기에서 합성한 cDNA 주형 2㎕, 10배 농축반응 완충용액 10㎕, 25mM MgCl26㎕, 10mM dNTP 2㎕ 0.75㎍ NdeIL-11 프라이머 3㎕, 0.75㎍ IL-11Sal 프라이머 3㎕, Taq 중합효소 0.4㎖(5U/㎖, Promega, U.S.A.)에 75㎕의 증류수를 넣어 반응액을 만들고 94℃, 1분 30초: 50℃, 1분; 72℃, 1분 30초의 반응을 40회 반복하여 562 염기쌍의 인간 인터루킨-11 유전자를 증폭하였다.Polymerase chain reaction was carried out using the cDNA strand as a template, except that 2 μl of the synthesized cDNA template, 10 μl of 10-fold concentrated buffer solution, 6 μl of 25 mM MgCl 2 , 2 μl of 10 mM dNTP 0.7 μg NdeIL-11 primer 3 μl , 75 μl of distilled water was added to 3 μl of 0.75 μg IL-11Sal primer and 0.4 mL of Taq polymerase (5 U / mL, Promega, USA) to make a reaction solution. 94 ° C., 1 minute 30 seconds: 50 ° C., 1 minute; The reaction at 40 DEG C and 1 minute 30 seconds was repeated 40 times to amplify the human interleukin-11 gene of 562 base pairs.
클리나우 중합효소(Klenow polymerase)와 T7 폴리뉴클레오티드 키나제(polynucleotide kinase, BRL, U.S.A.)를 처리하여 유전자의 양말단을 평활말단(blunt end)으로 만들어, HincII 제한효소로 절단된 pUC18 백터에 접합하였다(pUC18-IL-11). 대장균 JM105(ATCC 47016) 컴피턴트 세포에 접합 반응액을 첨가하여 주고 하나한의 방법(J. Mol. Biol. 116, 557(1983))에 따라 형질전환시킨 후, LB-엠피실린(50㎍/㎖ 앰피실린을 포함하는 LB 배지) 플레이트에 평판배양하여 대장균 형질전환체를 선별하였다. 생거 등의 방법(Sanger, F. et al., Proc. Natl. Acad. Sci. U.S.A. 74, 5463(1977)))에 따라 인간 인터루킨-11 유전자의 전체 핵산 염기서열을 확인하여 알려진 인간 인터루킨-11의 염기서열(Paul S. R. et al., Proc. Natl. Acad, Sci. U.S.A. 87, 7512(1990))과 동일함을 확인하였다.Klenow polymerase and T7 polynucleotide kinase (BRL, USA) were used to make the blunt end of the gene into a blunt end, which was then conjugated to a pUC18 vector digested with HincII restriction enzymes. pUC18-IL-11). Conjugated reaction solution was added to E. coli JM105 (ATCC 47016) competent cells and transformed according to one method (J. Mol. Biol. 116, 557 (1983)), followed by LB-Epicillin (50 μg / E. coli transformants were selected by plating on LB medium) plates containing ml ampicillin). According to Sanger et al. (Sanger, F. et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)), the entire nucleic acid sequence of the human interleukin-11 gene was identified and known human interleukin-11. It was confirmed that the nucleotide sequence of (Paul SR et al., Proc. Natl. Acad, Sci. USA 87, 7512 (1990)).
(단계 2) 인터루킨-11 유전자의 대장균 세포내 발현(Step 2) E. coli Intracellular Expression of Interleukin-11 Gene
본 출원인이 선출원(대한민국 특허출원 제 91-1360 호)에서의 발현벡터pTrpH-UB-CORE14(ATCC 68642) 2㎍을 제한효소 Ndel과 SelI으로 완전절단하고, 0.7% 아가로즈 겔로 전기영동 분리하여 약 2.5kb의 핵산절편을 얻었다. 이하,이 절편을 절편 NL이라 칭한다. 한편 단계 1에서 염기서열이 확인된 플라스미드 pUC18-IL-11을 제한효소 NdeI과 SalI으로 완전절단하고 545 염기쌍의 인터루킨-11 유전자 핵산절편을 분리하였다.Applicant completely cleaved 2 μg of the expression vector pTrpH-UB-CORE14 (ATCC 68642) from the previous application (Korean Patent Application No. 91-1360) with restriction enzymes Ndel and SelI, followed by electrophoresis separation with 0.7% agarose gel. A 2.5 kb nucleic acid fragment was obtained. Hereinafter, this intercept is referred to as intercept NL. Meanwhile, in step 1, the plasmid pUC18-IL-11, whose nucleotide sequence was identified, was completely cleaved with restriction enzymes NdeI and SalI, and 545 base pair interleukin-11 gene nucleic acid fragments were isolated.
인터루킨-11 핵산절편과 절편 NL을 다음과 같이 연결반응시켰다. 100ng의 절편 NL, 100ng의 인터루킨-11 절편, 2㎕의 10배 농도 연결반응 완충용액(500mM Tris-HCl, pH 7.8, 100mM MgCl2, 100mM ATP, 250㎍/㎖ BSA), 10단위의 T4 핵산 리가제를 넣고 총부피가 20㎕가 되도록 증류수를 가한 다음 16℃에서 12 시간 동안 반응시켰다. 이 반응액을 대장군 W3110(ATCC 37339)에 형질전환시킨 후, 50㎍/㎖의 앰피실린이 함유된 액체 루리아(Luria) 배지(6% 박토트립톤, 0.5% 효모 추출물, 1% 염화 나트륨)에서 12 시간 동안 진탕배양한 다음, 이중 100㎕을 2㎖의 M9 배지(40mM K2HPO4, 22mM KH2PO4, 8.5mM NaCl, 18.7mM NH4Cl, 1% 포도당, 0.1mM MgSO4, 0.1mM CaCl2, 0.4% 카사미노산, 10㎍/㎖ 비타민 B1, 40㎍/㎖ 앰피실린)로 옮겨서 37℃에서 약 4 시간 동안 진탕배양하였다. 박테리아 배양액의 흡광도가 650nm에서 약 0.3 정도가 될 때 인돌아크릴산(Indole Acylic Acid, IAA)을 최종농도가 50㎍/㎖이 되게 첨가하였다. IAA를 첨가한지 약 4 시간 후에 세포 배양액의 흡광도를 측정한 다음 원심분리기(Beckman J2-21, JA14 rotor)를 이용하여 11,000rpm에서 25 분간 원심분리하여 대장균 세포 침전물을 수거하였다. 수거한 10㎕의 대장균 추출물을램리의 방법(Laemmli, et al., Nature, 227, 680(1970))에 따라 15% SDS-폴리아크릴아미드 겔 전기영동시킨 후 코마시블루(Coomassie brilliant blue R250)로 단백질을 염색하였으나, 약 23,000 달톤 크기에서 음성시료에 비해 차이가 나는 단백질을 찾을 수가 없었다.The interleukin-11 nucleic acid fragment and the fragment NL were linked as follows. 100 ng fragment NL, 100 ng interleukin-11 fragment, 2 μl of 10-fold concentration ligation buffer (500 mM Tris-HCl, pH 7.8, 100 mM MgCl 2 , 100 mM ATP, 250 μg / ml BSA), 10 units of T4 nucleic acid Ligase was added and distilled water was added so that the total volume was 20 μl, and the reaction was performed at 16 ° C. for 12 hours. The reaction solution was transformed into colon W3110 (ATCC 37339) and then in liquid Luria medium (6% bactotriptone, 0.5% yeast extract, 1% sodium chloride) containing 50 μg / ml ampicillin. After shaking for 12 hours, 100 μl of 2 ml of M9 medium (40 mM K 2 HPO 4 , 22 mM KH 2 PO 4 , 8.5 mM NaCl, 18.7 mM NH 4 Cl, 1% glucose, 0.1 mM MgSO 4 , 0.1 mM CaCl 2 , 0.4% casamino acid, 10 μg / ml Vitamin B1, 40 μg / ml ampicillin) and shaken at 37 ° C. for about 4 hours. When the absorbance of the bacterial culture was about 0.3 at 650 nm, Indole Acylic Acid (IAA) was added to a final concentration of 50 µg / ml. About 4 hours after the addition of IAA, the absorbance of the cell culture was measured, and the E. coli cell precipitate was collected by centrifugation at 11,000 rpm for 25 minutes using a centrifuge (Beckman J2-21, JA14 rotor). Collected 10 μl of E. coli extracts were subjected to 15% SDS-polyacrylamide gel electrophoresis according to the method of Laemmli (Laemmli, et al., Nature, 227, 680 (1970)), followed by Coomassie brilliant blue R250. The protein was stained with, but it was not possible to find a protein that differs from the negative sample at about 23,000 Daltons.
(단계 3) 인터루킨-11 유전자의 효모세포내 발현(Step 3) Expression in Yeast Cells of Interleukin-11 Gene
인터루킨-11 유전자가 유비퀴틴과 융합되어 아미노 말단이 유비퀴틴 분해효소에 의해 절단될 수 있도록, 전구서열의 일부인 알라닌이 첨가되고 SacII 인지부위와 유비퀴틴 카복시 말단 일부가 포함된 프라이머를 합성하였다.In order for the interleukin-11 gene to be fused with ubiquitin and the amino terminus was cleaved by ubiquitin degrading enzyme, alanine, which is a part of the precursor sequence, was added, and a primer including a SacII recognition site and a part of the ubiquitin carboxy terminus was synthesized.
번째에 GCT(인터루킨-11의 전구서열중 마지막 아미노산인 알라닌을 코드)가 첨가되고 19번째 핵산부터 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안되었다.GCT (an alanine, which is the last amino acid in the precursor sequence of interleukin-11) was added to the gene and designed to have the same base sequence as the interleukin-11 gene from the 19th nucleic acid.
pUC18 IL-11 DNA를 주형으로 하여 중합효소 연쇄반응을 수행하였는데, 10배 농축반응 완충용액 10㎕, 25mM MgCl26㎕, 10mM dNTP 2㎕, 0.75㎍ T2IL-11 프라이머 3㎕, 0.75㎍ IL-IISal 프라이머 3㎕, Taq 중합효소 0.4㎕(5U/㎖, Promega, U.S.A.)에 75㎕의 증류수를 넣어 반응액을 만들고 94℃, 1분; 50℃, 1분; 72℃, 1분의 반응을 25회 반복하여 566 염기쌍의 인터루킨-11 유전자를 증폭하였다.The polymerase chain reaction was carried out using pUC18 IL-11 DNA as a template. 10 μl of 10-fold concentrated buffer solution, 6 μl of 25 mM MgCl 2 , 2 μl of 10 mM dNTP, 3 μl of 0.75 μg T2IL-11 primer, 0.75 μg IL- 75 µl of distilled water was added to 3 µl of IISal primer and 0.4 µl of Taq polymerase (5U / ml, Promega, USA) to make a reaction solution at 94 ° C. for 1 minute; 50 ° C., 1 minute; The reaction of 25 minutes at 72 ° C. was repeated 25 times to amplify the 566 base-pair interleukin-11 gene.
본 출원인의 선출원(대한민국 특허출원 제 89-20200 호)에서의 효모용 발현벡터 pYLBC A/G UB-HGH(ATCC 74071) 2㎍을 제한효소 PstI과 SalI으로 완전절단하고, 또한 동일 플라스미드 2㎍을 제한효소 PstI과 SacII으로 완전절단한 다음 0.7% 아가로즈 겔로 전기영동 분리하여 각각 9.8kb와 4.5kb 핵산절편을 얻었다. 이하, 이들 절편을 각각 절편 PL 및 절편 PT라 칭한다. 한편 상기에서 증폭된 인터루킨-11 핵산을 제한효소 SacII와 SalI으로 완전절단하고 7% 폴리아크릴아미드 겔 전기영동하여 540 염기쌍의 인터루킨-11 유전자 핵산절편을 분리하였다. 인터루킨-11 핵산절편과 절편 PL 및 절편 PT를 T4 핵산 리가제를 이용하여 연결반응시켰다. 이 반응액을 대장균 HB101(ATCC 33694) 컴피턴트 세포에 첨가하여 주고 형질전환시킨 후 LB-앰피실린(50㎍/㎖ 앰피실린을 포함하는 LB 배지) 플레이트에 평판배양하여 대장균 형질전환체를 선별하였다. 재조합 플라스미드 pYLBC A/G UB IL-11는 알칼리 용해방법(Alkaline lysis method; Ish, Horowicz. C. et al., Nucleic Acid Res. 9, 2989(1981))으로 대량추출하여 효모 형질전환에 이용하였다.2 μg of the yeast expression vector pYLBC A / G UB-HGH (ATCC 74071) in the applicant's prior application (Korean Patent Application No. 89-20200) was completely cut with restriction enzymes PstI and SalI, and 2 μg of the same plasmid After complete cleavage with restriction enzymes PstI and SacII, electrophoresis separation was performed with 0.7% agarose gel to obtain 9.8 kb and 4.5 kb nucleic acid fragments, respectively. Hereinafter, these fragments are called fragment PL and fragment PT, respectively. Meanwhile, the amplified interleukin-11 nucleic acid was completely cleaved with restriction enzymes SacII and SalI and subjected to 7% polyacrylamide gel electrophoresis to separate 540 base pairs of interleukin-11 gene nucleic acid fragments. Interleukin-11 nucleic acid fragments, fragment PL and fragment PT were ligated using T4 nucleic acid ligase. The reaction solution was added to E. coli HB101 (ATCC 33694) competent cells, transformed, and plated on LB-ampicillin (LB medium containing 50 µg / ml ampicillin) to select E. coli transformants. . Recombinant plasmid pYLBC A / G UB IL-11 was used for yeast transformation by mass extraction with alkaline lysis method (Ikaline lysis method; Ish, Horowicz. C. et al., Nucleic Acid Res. 9, 2989 (1981)). .
이어서, 베그(Beggs, et al., Nature, 275, 104(1978))와 힌넨등의 방법(Hinnen, et al., Proc. Natl. Acad. Sci. U.S.A. 75, 1929(1978))을 기본으로 하여 다음과 같이 효모를 형질전환시켰다. 즉, 효모(Saccharomyces cerevisiae DC04, Yeast Genetic Stoock Center, Univ. of California, Berkeley CA., U.S.A.)를 YEPD 배지(펩톤 2%, 효모 추출물 1%, 글루코스 2%)에 접종하여 흡광도가 0.8 내지 1.0이 될 때까지 30℃에서 진탕배양하였다. 상기 세포 침전물을 물과 1M 솔비톨 용액으로 씻은 후 원심분리하였다. 침전된 세포를 5㎖의 SP 완충용액(1M 솔비톨, 50mM 인산칼륨, pH 7.5), 5㎕의 1M 디티오스레이톨(DTT)에 잘 녹인 후, 50㎕의 자이몰라제(Zymolase, 10mg/㎖ in 50% 글리세롤/50% SP 완충용액)를 넣어 주고 30℃의 완만한 진탕조건(150rpm)에서 30분 정도 반응시켜 스페로플라스트를 얻은 후 5㎖의 1M 솔비톨 및 5㎖의 STC 완충용액(1M 솔비톨, 10mM Tris-Cl, pH 7.5)으로 두차례 씻어주고 최종 0.5㎖의 STC 완충용액에 녹여 형질전환에 이용하였다. 12.5㎕의 10㎍ 재조합 플라스미드 pYLBC A/G UB IL-11과 12.5㎕의 2×STC(STC의 2배 농축액)를 잘 섞은 후 상기의 스페로플라스트 50㎕를 첨가하고, 상온에서 10 분간 배양하였다. 50㎕의 PEG/TC 용액(40% PEG-4000, 10mM CaCl, 10mM Tris-HCl, pH 7.5)을 첨가한 후 10 분을 더 방치시킨 후 1,500rpm에서 3 분간 원심분리하여 스페로플라스트 침전을 얻었고, 이것을 200㎕의 1M 솔비톨에 잘 녹였다. 7㎖의 재생아가(Regeneration Agar: 100㎖당 3g 박토아가, 50㎖의 2M 솔비톨, 0.07g의 아미노산 없는 효모 질소기질, 루이신이 결핍된 0.1g의 아미노산 혼합물, 0.4㎖의 50% 글루코스, 45㎖의 물)와 혼합하여 Leu-/솔비톨 플레이트(루이신이 결핍된 플레이트)에 도포한 후 30℃에서 5 일간 배양하여 재조합 플라스미드에 의해 형질전환된 효모 콜로니를 얻었다.Then, based on Beggs, et al., Nature, 275, 104 (1978) and Hinnen et al. (Hinnen, et al., Proc. Natl. Acad. Sci. USA 75, 1929 (1978)). Yeast was transformed as follows. In other words, yeast (Saccharomyces cerevisiae DC04, Yeast Genetic Stoock Center, Univ. Of California, Berkeley CA., USA) was inoculated in YEPD medium (peptone 2%, yeast extract 1%, glucose 2%) absorbance 0.8 to 1.0 Shake culture at 30 ℃ until. The cell precipitate was washed with water and 1M sorbitol solution and then centrifuged. Precipitated cells were dissolved in 5 ml of SP buffer (1 M sorbitol, 50 mM potassium phosphate, pH 7.5), 5 µl of 1 M dithiositol (DTT), and then 50 µl of Zymolase (10 mg / ml). in 50% glycerol / 50% SP buffer solution and reacted for 30 minutes under gentle shaking condition of 30 ℃ (150rpm) to obtain spheroplast, 5ml of 1M sorbitol and 5ml of STC buffer (1M Sorbitol, 10mM Tris-Cl, pH 7.5) was washed twice and dissolved in the final 0.5ml STC buffer and used for transformation. After mixing 12.5 μl of 10 μg recombinant plasmid pYLBC A / G UB IL-11 and 12.5 μl of 2 × STC (double concentration of STC), 50 μl of the above spheroplast was added and incubated for 10 minutes at room temperature. . 50 μl of PEG / TC solution (40% PEG-4000, 10 mM CaCl, 10 mM Tris-HCl, pH 7.5) was added and then left for 10 minutes, followed by centrifugation at 1,500 rpm for 3 minutes to obtain spheroplast precipitation. This was dissolved in 200 µl of 1M sorbitol. 7 ml Regeneration Agar (3 g bactoa per 100 ml, 50 ml 2M sorbitol, 0.07 g yeast nitrogen substrate without amino acid, 0.1 g amino acid mixture lacking leucine, 0.4 ml 50% glucose, 45 ml Water), and applied to a Leu − / sorbitol plate (a plate lacking leucine) and incubated at 30 ° C. for 5 days to obtain a yeast colony transformed with the recombinant plasmid.
형질전환된 효모 균주를 3㎖의 루이신(leucine)이 결핍된 배양액(배양액 1ℓ당 아미노산이 없는 효모 질소기질(yeast nitrogen base without amino acids, Difco, U.S.A.) 6.7g과 루이신이 결핍된 아미노산 혼합물 0.25g 및 5% 글루코스)에 접종하여 30℃에서 24 시간 동안 배양하고 15,000rpm에서 3 분간 원심분리하여 효모세포 농축액을 얻은 다음, 2% 글루코스가 함유된 YEP 배지(펩톤 2%, 효모추출물 1%)에 현탁시켜 48 시간 배양하였다. 각각의 배양액을 원심분리하여 400㎕의 완충용액(10mM Tris-HCl, pH 7.5, 1mM PMSF(Phenylmethylsulfonyl fluoride), 8M Urea)에 현탁하고 직경 0.4mm의 유리구슬을 같은 부피로 첨가하여 강하게 진탕시켜 세포가 파괴된 효모 추출물을 얻었다. 10㎕의 효모추출물을 램리의 방법(Laemmli, et al., Nature 227, 680(1970))에 따라 15% SDS-폴리아크릴아미드 겔 전기영동시킨 후 코마시블루(Coomassie brilliant blue R250)로 단백질을 염색하였으나, 약 23,000 달톤 크기에서 음성시료에 비해 차이가 나는 단백질을 찾을 수가 없었다.6.7 g of the transformed yeast strain was cultured lacking 3 ml of leucine (yeast nitrogen base without amino acids, Difco, USA) and amino acid mixture lacking leucine 0.25 g and 5% glucose), incubated for 24 hours at 30 ° C., and centrifuged at 15,000 rpm for 3 minutes to obtain yeast cell concentrate, followed by YEP medium containing 2% glucose (2% peptone and 1% yeast extract). Suspended and incubated for 48 hours. Each culture solution was centrifuged and suspended in 400 µl of buffer solution (10 mM Tris-HCl, pH 7.5, 1 mM PMSF (Phenylmethylsulfonyl fluoride), 8 M Urea), and the glass beads having a diameter of 0.4 mm were added to the same volume and shaken strongly. The yeast extract was destroyed. 10 μl of the yeast extract was subjected to 15% SDS-polyacrylamide gel electrophoresis according to the method of Laymmli, et al., Nature 227, 680 (1970), followed by protein extraction with Coomassie brilliant blue R250. Staining, however, could not find a protein that differs from the negative sample at about 23,000 Daltons.
실 시 예 : 변형된 인간 rIL-11 유전자의 발현Example: Expression of Modified Human rIL-11 Gene
(단계 1) rIL-11 유전자의 고안 합성 및 클로닝(Step 1) Design Synthesis and Cloning of rIL-11 Gene
인터루킨-11 유전자의 염기서열을 아미노산의 변화가 없도록 하면서 효모에서 선호하는 염기코돈으로 아래와 같이 변형된 재조합 rIL-11 유전자를 고안하였다:The recombinant rIL-11 gene was modified with the following preferred base codons in yeast without altering the nucleotide sequence of the interleukin-11 gene:
상기 rIL-11 유전자의 클로닝을 위해 5'-말단에 NdeI 인지부위와 메티오닌 번역시작 코돈을 갖고, 또한 3'-말단에 종료코돈 TAG와 SalI 인지부위를 갖는 핵산서열이 첨가된 전체 558 염기쌍의 유전자를 만들기 위해 12 개의 올리고뉴클레오티드를 합성하였다.For cloning the rIL-11 gene, a gene having a total of 558 base pairs having a NdeI recognition site and a methionine translation start codon at the 5'-end and a nucleic acid sequence having the end codon TAG and SalI recognition site at the 3'-end was added 12 oligonucleotides were synthesized to make.
(밑줄)를 포함하고 12번째 핵산부터 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안되었고;(Underscore) and have the same base sequence as the interleukin-11 gene from the twelfth nucleic acid;
자와 동일한 염기서열을 갖도록 고안되었고;Is designed to have the same nucleotide sequence as the ruler;
CCACTA-5')는 77번째 핵산부터 144번째 핵산까지 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안되었고;CCACTA-5 ') is designed to have the same base sequence as the interleukin-11 gene from the 77 th nucleic acid to the 144 th nucleic acid;
GAGATGTT-5')는 136번째 핵산부터 204번째 핵산까지 인터루킨-11 유전자와 동일한 염기서열를 갖도록 고안되었고;GAGATGTT-5 ') is designed to have the same base sequence as the interleukin-11 gene from the 136th nucleic acid to the 204th nucleic acid;
산부터 240번째 핵산까지 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안되었고;Designed to have the same nucleotide sequence as the interleukin-11 gene from acid to 240 th nucleic acid;
산부터 280번째 핵산까지 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안되었고;Designed to have the same base sequence as the interleukin-11 gene from acid to 280th nucleic acid;
번째 핵산부터 325번째 핵산까지 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안되었고;Designed to have the same base sequence as the interleukin-11 gene from the first nucleic acid to the 325 th nucleic acid;
ACCG-5')는 315번째 핵산부터 380번째 핵산까지 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안되었고;ACCG-5 ') is designed to have the same base sequence as the interleukin-11 gene from the 315th nucleic acid to the 380th nucleic acid;
째 핵산부터 420번째 핵산까지 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안되었고;Designed to have the same nucleotide sequence as the interleukin-11 gene from the first nucleic acid to the 420th nucleic acid;
째 핵산부터 463번째 핵산까지 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안되었고;Designed to have the same nucleotide sequence as the interleukin-11 gene from the first nucleic acid to the 463th nucleic acid;
산부터 503번째 핵산까지 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안되었고;Designed to have the same base sequence as the interleukin-11 gene from acid to 503 nucleic acid;
료코돈과 SalI 인지부위(밑줄)를 포함하며 485번째 핵산부터 534번째 핵산까지 인터루킨-11 유전자와 동일한 염기서열을 갖도록 DNA 합성기를 이용하여 고안합성하였다.The DNA synthesizer was designed to include the lycodone and SalI recognition sites (underlined) and have the same base sequence as the interleukin-11 gene from the 485 th nucleic acid to the 534 th nucleic acid.
이어서 다음과 같이 중합효소 연쇄반응을 수행하여 전체 유전자를 연결하였다; 먼저 10배 농축반응 완충용액 10㎕, 25mM MgCl26㎕, 10mM dNTP 2㎕, 0.75㎍ IL-11C1 프라이머 3㎕, 0.75㎍ IL-11C4 프라이머 3㎕, IL-11C2 프라이머 20ng, IL-11C3 프라이머 20ng, Taq 중합효소 0.4㎕(5U/㎖, Promega, U.S.A.)에 75㎕의 증류수를 넣어 반응액을 만들고 94℃, 1분; 50℃, 1분: 72℃, 1분 30초의 반응을 25회 반복하여 215 염기쌍의 N-말단 일부 인터루킨-11 유전자(C 절편)를 증폭하였다. 동일한 조건하에서 IL-11D1 프라이머 0.75㎍, IL-11D4 프라이머 0.75㎍, IL-11D2 프라이머 20ng, IL-11D3 프라이머 20ng을 이용하여 192 염기쌍의 일부 인터루킨-11 유전자(D 절편)를 증폭하였다. 또한 IL-11E1 프라이머 0.75㎍, IL-11E4 프라이머 0.75㎍, IL-11E2 프라이머 20ng, IL-11E3 프라이머 20ng을 이용하여 183 염기쌍의 C-말단 일부 인터루킨-11 유전자(E 절편)를 증폭하였다.Subsequently, the polymerase chain reaction was performed to link the entire genes as follows; First 10 fold concentrated buffer 10 μl, 25 mM MgCl 2 6 μl, 10 mM dNTP 2 μl, 0.75 μg IL-11C1 primer 3 μl, 0.75 μg IL-11C4 primer 3 μl, IL-11C2 primer 20ng, IL-11C3 primer 20ng , 0.4 μl of Taq polymerase (5 U / mL, Promega, USA) was added to 75 μl of distilled water to make a reaction solution, 94 ° C. for 1 minute; The reaction of 50 ° C., 1 minute: 72 ° C., 1 minute 30 seconds was repeated 25 times to amplify the N-terminal part Interleukin-11 gene (C fragment) of 215 base pairs. Under the same conditions, some interleukin-11 gene (D fragment) of 192 base pairs was amplified using 0.75 μg of IL-11D1 primer, 0.75 μg of IL-11D4 primer, 20 ng of IL-11D2 primer, and 20 ng of IL-11D3 primer. Furthermore, 183 base-pair C-terminal part interleukin-11 gene (E fragment) was amplified using 0.75 μg of IL-11E1 primer, 0.75 μg of IL-11E4 primer, 20ng of IL-11E2 primer, and 20ng of IL-11E3 primer.
C, D 및 E 절편을 주형으로 하여 IL-11C1 프라이머와 IL-11E4 프라이머를 이용하여 중합효소 연쇄반응을 수행하여 558 염기쌍의 전체 유전자를 합성한 다음,이를 클리나우 중합효소(Klenow polymerase)와 T7 폴리뉴클레오티드 키나제(polynucleotide kinase, BRL, U.S.A.)를 처리하고 HincII 제한효소로 절단된 pUC18 벡터에 접합하였다(pUC18-rIL-11). 대장균 JM105(ATCC 47016) 컴피턴트 세포에 접합 반응액을 첨가하여 주고 하나한의 방법(J. Mol. Biol. 116, 557(1983))에 따라 상기 발현벡터로 형질전환시킨 후, LB-앰피실린(50㎍/㎖ 앰피실린을 포함하는 LB 배지) 플레이트에 평판배양하여 대장균 형질전환체를 선별하였다. 인간 rIL-11 유전자의 전체 핵산 염기서열이 시퀀싱을 통해 상기에서 고안한 인간 rIL-11의 염기서열과 동일함을 확인하였다(제 1 도).Using the C, D, and E fragments as templates, a polymerase chain reaction was carried out using IL-11C1 primers and IL-11E4 primers to synthesize 558 base pairs of all genes, followed by Klenow polymerase and T7. Polynucleotide kinase (BRL, USA) was treated and conjugated to pUC18 vector digested with HincII restriction enzyme (pUC18-rIL-11). Conjugated reaction solution was added to E. coli JM105 (ATCC 47016) competent cells and transformed with the expression vector according to one method (J. Mol. Biol. 116, 557 (1983)), followed by LB-ampicillin. E. coli transformants were selected by plating on plates (LB medium containing 50 μg / ml ampicillin). Through sequencing, the entire nucleic acid sequence of the human rIL-11 gene was confirmed to be identical to the nucleotide sequence of the human rIL-11 designed above (FIG. 1).
(단계 2) 효모 발현벡터 pYLBC A/G UB rIL-11의 클로닝 및 IL-11 단백질의 발현(Step 2) Cloning of yeast expression vector pYLBC A / G UB rIL-11 and expression of IL-11 protein
rIL-11 유전자의 발현을 높이기 위해, 변형된 rIL-11 유전자의 아미노 말단이 유비퀴틴 분해효소에 의해 절단될 수 있도록, 전구서열의 일부인 알라닌이 첨가되고 SacII 인지부위와 유비퀴틴 카복시 말단 일부를 포함하는 T2AlaIL-11 프라이머를 합성하였다.To increase the expression of the rIL-11 gene, T2AlaIL containing a SacII recognition site and a portion of the ubiquitin carboxy terminus is added to alanine, which is part of the precursor sequence, so that the amino terminus of the modified rIL-11 gene can be cleaved by ubiquitin degrading enzymes. -11 primers were synthesized.
18 번째에 GCT(IL-11의 전구서열중 마지막 아미노산인 알라닌을 코드)가 첨가되고 19번째 핵산부터 인터루킨-11 유전자와 동일한 염기서열을 갖도록 고안하였다. pUC18-rIL-11 DNA를 주형으로 하여 중합효소 연쇄반응을 수행하였는데, 10배 농축반응 완충용액 10㎕, 25mM MgCl26㎕, 10mM dNTP 2㎕, 0.75㎍ T2AlaIL-11 프라이머 3㎕, 0.75㎍ IL-11E4 프라이머 3㎕, Taq 중합효소 0.4㎕(5U/㎖, Promegs, U.S.A.)에 75㎕의 증류수를 넣어 반응액을 만들고 94℃, 1분; 50℃, 1분; 72℃, 1분의 반응을 25회 반복하여 566 염기쌍의 rIL-11 유전자를 증폭하였다.At the 18th, GCT (an alanine, which is the last amino acid in the precursor sequence of IL-11) was added and designed to have the same base sequence as the interleukin-11 gene from the 19th nucleic acid. The polymerase chain reaction was carried out using pUC18-rIL-11 DNA as a template. 10 μl of 10 times concentrated buffer solution, 6 μl of 25 mM MgCl 2 , 2 μl of 10 mM dNTP, 3 μl of 0.75 μg T2AlaIL-11 primer, 0.75 μg IL 3 µl of 11E4 primer and 0.4 µl of Taq polymerase (5 U / ml, Promegs, USA) were added to 75 µl of distilled water to make a reaction solution at 94 ° C. for 1 minute; 50 ° C., 1 minute; The reaction of 25 minutes at 72 ° C. was repeated 25 times to amplify the rIL-11 gene of 566 base pairs.
비교예의 단계 3에서와 같은 방식으로 rIL-11 유전자를 제한효소 SacII 및 SalI으로 완전절단하고 7% 폴리아크릴아미드 겔 전기영동하여 분리한 핵산절편을 pYLBC A/G UB-HGH 유래 절편 PL 및 PT와 T4 핵산 리가제를 이용하여 연결반응시켰다. 이 반응액을 대장균 HB101(ATCC 33694) 컴피턴트 세포에 첨가하여 주고 형질전환시킨 후, LB-앰피실린(50㎍/㎖ 앰피실린을 포함하는 LB 배지) 플레이트에 평판배양하여 대장균 형질전환체를 선별하였다.In the same manner as in step 3 of the comparative example, nucleic acid fragments which were completely cleaved with restriction enzymes SacII and SalI and isolated by 7% polyacrylamide gel electrophoresis were isolated from pYLBC A / G UB-HGH-derived fragments PL and PT. The ligation was performed using T4 nucleic acid ligase. The reaction solution was added to E. coli HB101 (ATCC 33694) competent cells and transformed, followed by plate culture on LB-ampicillin (LB medium containing 50 µg / ml ampicillin) to screen for E. coli transformants. It was.
제 2 도는 본 발명에 따라 변형된 인간 인터루킨-11 유전자를 효모 발현벡터 pYLBC A/G UB rIL-Il으로 클로닝하는 과정을 도시한 것이다.Figure 2 shows the procedure for cloning the human interleukin-11 gene modified according to the present invention into the yeast expression vector pYLBC A / G UB rIL-Il.
재조합 플라스미드 pYLBC A/G UB rIL-11을 알칼리 용해 방법으로 대량추출하여 상기 비교예 단계 3과 동일한 방법으로 사카로마 이세스 세레비지애 DC04에 형질전환시켜 재조합 효모 콜로니를 얻었다.Recombinant plasmid pYLBC A / G UB rIL-11 was extracted in a large amount by an alkali lysis method and transformed into Saccharomyces cerevisiae DC04 in the same manner as in Comparative Example 3 to obtain a recombinant yeast colony.
상기 형질전환된 사카로마이세스 세레비지애 DC04/pYLBC A/G UB rIL-11을 1995년 12월 28일자로 한국과학기술연구원 생명공학연구소 부설 유전자은행에 기탁번호 제 KCTC 8721P 호로서 기탁하였다.The transformed Saccharomyces cerevisiae DC04 / pYLBC A / G UB rIL-11 was deposited on Dec. 28, 1995 to the gene bank attached to the Biotechnology Research Institute of Korea Institute of Science and Technology as Accession No. KCTC 8721P.
형질전환된 효모 균주를 2% 글루코스가 함유된 YEP 배지(펩톤 2%, 효모 추출물 1%)에 현탁시켜 48 시간 동안 배양한 후 상기와 동일한 방법으로 세포를 파괴하고 효모 추출물을 얻었다. 10㎕의 효모추출물을 램리의 방법(Laemmli, et al., Nature, 227, 680(1970))에 따라 15% SDS-폴리아크릴아미드 겔 전기영동시킨 후 코마시블루(Coomassie brilliant blue R250)로 단백질을 염색하였다.The transformed yeast strain was suspended in YEP medium containing 2% glucose (peptone 2%, yeast extract 1%), incubated for 48 hours, and then cells were destroyed in the same manner as above to obtain yeast extract. 10 μl of yeast extract was subjected to 15% SDS-polyacrylamide gel electrophoresis according to the method of Laemmli, et al., Nature, 227, 680 (1970), followed by proteinization with Coomassie brilliant blue R250. Was stained.
제 3 도는 본 발명의 발현벡터 pYLBC A/G UB rIL-11으로 형질전환된 효모를 배양한 후 변성 폴리아크릴아미드 겔 전기영동한 결과를 나타낸 것이다. 제 1 열은 벡터를 함유하지 않는 효모의 추출액으로 음성조건 시료이고, 제 2 열 및 제 3 열은 본 발명의 발현벡터 pYLBC A/G UB rIL-11으로 형질전환된 두개의 서로 다른 효모클론의 추출액 시료이고, 제 4 열은 표준 단백질 분자량을 표시하는 것이다.Figure 3 shows the result of modified polyacrylamide gel electrophoresis after culturing yeast transformed with the expression vector pYLBC A / G UB rIL-11 of the present invention. Column 1 is an extract of yeast that does not contain a vector and is a negative condition sample. Column 2 and column 3 are two different yeast clones transformed with the expression vector pYLBC A / G UB rIL-11 of the present invention. An extract sample, and the fourth column shows the standard protein molecular weight.
여기에서 보면, 약 23,000 달톤 크기에서 단일한 인간 인터루킨-11 단백질이 생성되었음을 확인할 수 있었다. 앞서 비교예에서 제조한 바와 같이, 종래의 cDNA 염기서열을 갖는 인터루킨-11 유전자를 유비퀴틴 유전자와 융합발현시킨 경우에는, 23,000 달톤 크기의 단백질생성이 없었던 것에 비추어 볼 때, 이러한 결과는 유비퀴틴에 의한 발현증가 효과에 의한 것이 아니라 변형된 합성 rIL-11 유전자의 차이에 기인한 것임을 알 수 있다.Here, it was confirmed that a single human interleukin-11 protein was produced at a size of about 23,000 Daltons. As prepared in the comparative example, when the interleukin-11 gene having the cDNA sequence was fused and expressed with the ubiquitin gene, the result was not expressed in the light of 23,000 Dalton-sized protein production. It can be seen that this is not due to the increasing effect but due to the difference in the modified synthetic rIL-11 gene.
제 1 도는 본 발명의 변경된 재조합 인간 인터루킨-11의 염기서열 및 아미노산 서열을 나타낸 것이고,Figure 1 shows the nucleotide sequence and amino acid sequence of the modified recombinant human interleukin-11 of the present invention,
제 2 도는 인간 인터루킨-11 유전자를 효모 발현벡터 pYLBC A/G-UB-rIL-11으로 클로닝하는 과정을 도시한 것이고,2 shows the cloning of the human interleukin-11 gene into the yeast expression vector pYLBC A / G-UB-rIL-11,
제 3 도는 본 발명의 발현벡터 pYLBC A/G-UB-rIL-11으로 형질전환된 효모세포를 배양한 후 변성 폴리아크릴아미드 겔 전기영동한 결과를 나타낸 것이다.Figure 3 shows the result of denatured polyacrylamide gel electrophoresis after culturing yeast cells transformed with the expression vector pYLBC A / G-UB-rIL-11 of the present invention.
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| JPS6091995A (en) * | 1983-10-18 | 1985-05-23 | Ajinomoto Co Inc | Preparation of interleukin-2 by yeast belonging to genus saccharomyces |
| US5215895A (en) * | 1989-11-22 | 1993-06-01 | Genetics Institute, Inc. | Dna encoding a mammalian cytokine, interleukin-11 |
| US5371193A (en) * | 1990-05-21 | 1994-12-06 | Genetics Institute, Inc. - Legal Affairs | Mammalian cytokine, IL-11 |
| JPH07224097A (en) * | 1994-02-08 | 1995-08-22 | Asahi Glass Co Ltd | Interleukin-6 variant |
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| JPS6091995A (en) * | 1983-10-18 | 1985-05-23 | Ajinomoto Co Inc | Preparation of interleukin-2 by yeast belonging to genus saccharomyces |
| US5215895A (en) * | 1989-11-22 | 1993-06-01 | Genetics Institute, Inc. | Dna encoding a mammalian cytokine, interleukin-11 |
| US5371193A (en) * | 1990-05-21 | 1994-12-06 | Genetics Institute, Inc. - Legal Affairs | Mammalian cytokine, IL-11 |
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