CN111748544B

CN111748544B - Method for efficiently expressing lysostaphin

Info

Publication number: CN111748544B
Application number: CN202010665840.5A
Authority: CN
Inventors: 梁伟凡; 李阳源; 唐雪梅; 容晓燕; 邓智远; 刘金山; 邓敬阳; 林影
Original assignee: Guangdong Vtr Bio Tech Co ltd
Current assignee: Guangdong Vtr Bio Tech Co ltd
Priority date: 2020-07-12
Filing date: 2020-07-12
Publication date: 2024-02-06
Anticipated expiration: 2040-07-12
Also published as: CN111748544A

Abstract

The invention relates to the field of genetic engineering, in particular to a method for efficiently expressing lysostaphin. The invention adopts a Pichia expression system to carry out secretion expression of lysostaphin, realizes mass expression of the lysostaphin, reduces the production cost, ensures that the highest expression level of the lysostaphin reaches 6g/L, and the lysostaphin is secreted and expressed in fermentation supernatant without breaking cells, and can obtain crude lysostaphin liquid with little impurity protein by simple filtration and removal of thalli.

Description

Method for efficiently expressing lysostaphin

Technical Field

The invention relates to the field of genetic engineering, in particular to a method for efficiently expressing lysostaphin.

Background

Staphylococcal infection is an infection caused by staphylococci. Lysostaphin (EC 3.4.24.75) is an endopeptidase that cleaves pentaglycine peptide bridge structures in cell wall peptidoglycans, thereby rapidly lysing bacteria. Because the cell wall of the staphylococcus is rich in pentaglycine peptide bridge structure, the lysostaphin has particularly remarkable sterilization effect on the staphylococcus, especially on drug-resistant staphylococcus aureus MRSA, and has certain antibacterial effect on other gram-positive bacteria. Lysostaphin hydrolyzes bacterial cell walls to prevent bacteria from growing normally, thereby achieving a sterilizing effect, and the unique antibacterial mechanism determines that the lysostaphin is not easy to generate drug resistance.

Lysostaphin is composed ofStaphylococcus simulansSecretory expression (mimicking staphylococci), but yields of natural origin are low and are themselves pathogenic and unsuitable for industrial fermentation production. With the development of genetic engineering techniques, recombinant expression of lysostaphin in a number of species has been achieved. Although the lysostaphin has realized heterologous recombinant expression and the expression level is greatly improved compared with a natural host, the expression level of the lysostaphin is still low as a whole, the expression level is not more than 0.5g/L, and the industrialized production cost is high.

Pichia pastoris expression systems are one of the most commonly used protein expression systems, but the processing folding process of the foreign protein after expression and translation in a host is difficult to predict, so whether the specific foreign protein can be efficiently expressed in the Pichia pastoris expression system still needs to overcome the technical problem which is difficult to predict.

Disclosure of Invention

The invention aims to provide a method for efficiently expressing lysostaphin.

It is a further object of the present invention to provide a method for biologically preparing lysostaphin.

The method for efficiently expressing the lysostaphin comprises the step of expressing an optimized lysostaphin encoding gene in pichia pastoris, wherein the nucleotide sequence of the optimized lysostaphin encoding gene is shown as SEQ ID NO.3, SEQ ID NO.12 or SEQ ID NO. 13.

A method for biologically preparing lysostaphin according to the present invention, said method comprising the steps of:

expressing an optimized lysostaphin encoding gene in pichia pastoris, wherein the nucleotide sequence of the optimized lysostaphin encoding gene is shown as SEQ ID NO.3, SEQ ID NO.12 or SEQ ID NO. 13;

separating lysostaphin in the fermentation supernatant.

Although the heterologous recombinant expression of the lysostaphin is realized, the expression quantity is improved compared with that of a natural production strain, the current expression level is still lower, and the production cost is higher. Meanwhile, the invention also provides a production method of the lysostaphin and a protein separation and purification method. The maximum expression level of lysostaphin of the Pichia pastoris expression system used in the invention reaches 6g/L, which is the highest level of public report. And the lysostaphin is secreted and expressed in the fermentation supernatant, cells are not required to be broken, the crude lysostaphin enzyme liquid with little impurity protein can be obtained by simply filtering and removing thalli, a high-purity lysostaphin purified sample can be obtained by only one-step ion exchange or affinity chromatography, and the cost of protein separation and purification is reduced. The lysostaphin sample prepared by the invention has obvious inhibition effect on staphylococcus, especially staphylococcus aureus.

Drawings

FIG. 1 shows the results of PCR electrophoresis verification of Top10/pPIczαA-Lss colonies, wherein M: standard nucleic acid molecular weight markers; 1-6: top10/pPICz alpha A-Lss colony PCR verification band;

fig. 2 shows the results of fermentation supernatant inhibition zone detection, wherein 1: optimization sequence 1 fermentation supernatant, 2: optimizing sequence 2 fermentation supernatant, 3: optimization sequence 3 fermentation supernatant, 4: primary sequence fermentation supernatant, 5: fermenting in a fermentation tank for 40h, and obtaining supernatant stock solution, and 6: fermenting in a fermentation tank for 110h, diluting the supernatant 5 times, and 7: fermenting for 180h in a fermentation tank, and diluting the supernatant by 10 times;

FIG. 3 is a fermentation curve of lysostaphin;

FIG. 4 shows the results of lysostaphin protein electrophoresis;

FIG. 5 shows the results of a lysostaphin thermotolerance assessment, wherein 1:50 ℃,2:60 ℃,3:70 ℃,4:80 ℃,5:90 ℃,6:100 ℃.

Description of the embodiments

The experimental procedures, which are not specified in the following examples, were carried out with reference to the conditions described in the molecular Cloning laboratory Manual (molecular Cloning: A Laboratory Manual, 2002), or according to the kit and instructions. DNA polymerase, restriction endonuclease, DNA ligase were purchased from New England Biolabs company, X33 Pichia pastoris and expression vectors thereof were purchased from Invitrogen company. Other materials are commercially available unless otherwise specified.

EXAMPLE 1 construction of lysostaphin-expressing Strain

Obtaining from NCBI databaseStaphylococcus simulansThe lysostaphin amino acid sequence (mimicking staphin) is shown in SEQ ID NO. 1. According to Pichia pastorisPichia pastoris) Codon preference (http:// www.kazusa.or.jp/codon/cgi-bin/showcode. Cgis species=4922) codon optimization is carried out on the mature region (the amino acid sequence is shown as SEQ ID NO.2, the gene sequence is shown as SEQ ID NO. 11) of the lysostaphin, and a termination codon TAA is added at the tail end of the gene sequence to obtain the mature lysostaphin gene sequence, wherein the codon optimization gene sequence is shown as SEQ ID NO.3, SEQ ID NO.12 and SEQ ID NO. 13.

Lysostaphin amino acid, SEQ ID NO.1

1 MKKTKNNYYT RPLAIGLSTF ALASIVYGGI QNETHASEKS NMDVSKKVAE

51 VETSKAPVEN TAEVETSKAP VENTAEVETS KAPVENTAEV ETSKAPVENT

101 AEVETSKAPV ENTAEVETSK APVENTAEVE TSKAPVENTA EVETSKAPVE

151 NTAEVETSKA PVENTAEVET SKAPVENTAE VETSKAPVEN TAEVETSKAP

201 VENTAEVETS KAPVENTAEV ETSKAPVENT AEVETSKALV QNRTALRAAT

251 HEHSAQWLNN YKKGYGYGPY PLGINGGMHY GVDFFMNIGT PVKAISSGKI

301 VEAGWSNYGG GNQIGLIEND GVHRQWYMHL SKYNVKVGDY VKAGQIIGWS

351 GSTGYSTAPH LHFQRMVNSF SNSTAQDPMP FLKSAGYGKA GGTVTPTPNT

401 GWKTNKYGTL YKSESASFTP NTDIITRTTG PFRSMPQSGV LKAGQTIHYD

451 EVMKQDGHVW VGYTGNSGQR IYLPVRTWNK STNTLGVLWG TIK 。

Mature region of lysostaphin, SEQ ID NO.2

1 AATHEHSAQW LNNYKKGYGY GPYPLGINGG MHYGVDFFMN IGTPVKAISS

51 GKIVEAGWSN YGGGNQIGLI ENDGVHRQWY MHLSKYNVKV GDYVKAGQII

101 GWSGSTGYST APHLHFQRMV NSFSNSTAQD PMPFLKSAGY GKAGGTVTPT

151 PNTGWKTNKY GTLYKSESAS FTPNTDIITR TTGPFRSMPQ SGVLKAGQTI

201 HYDEVMKQDG HVWVGYTGNS GQRIYLPVRT WNKSTNTLGV LWGTIK 。

Codon optimized gene sequence: SEQ ID NO.3

1 GCTGCTACTC ACGAACACTC TGCTCAATGG TTGAACAACT ACAAGAAGGG

51 TTACGGTTAC GGTCCATACC CATTGGGTAT CAACGGTGGT ATGCACTACG

101 GTGTTGACTT CTTCATGAAC ATCGGTACTC CAGTTAAGGC TATCTCTTCT

151 GGTAAGATCG TTGAAGCTGG TTGGTCTAAC TACGGTGGTG GTAACCAAAT

201 CGGTTTGATC GAAAACGACG GTGTTCACAG ACAATGGTAC ATGCACTTGT

251 CTAAGTACAA CGTTAAGGTT GGTGACTACG TTAAGGCTGG TCAAATCATC

301 GGTTGGTCTG GTTCTACTGG TTACTCTACT GCTCCACACT TGCACTTCCA

351 AAGAATGGTT AACTCTTTCT CTAACTCTAC TGCTCAAGAC CCAATGCCAT

401 TCTTGAAGTC TGCTGGTTAC GGTAAGGCTG GTGGTACTGT TACTCCAACT

451 CCAAACACTG GTTGGAAGAC TAACAAGTAC GGTACTTTGT ACAAGTCTGA

501 ATCTGCTTCT TTCACTCCAA ACACTGACAT CATCACTAGA ACTACTGGTC

551 CATTCAGATC TATGCCACAA TCTGGTGTTT TGAAGGCTGG TCAAACTATC

601 CACTACGACG AAGTTATGAA GCAAGACGGT CACGTTTGGG TTGGTTACAC

651 TGGTAACTCT GGTCAAAGAA TCTACTTGCC AGTTAGAACT TGGAACAAGT

701 CTACTAACAC TTTGGGTGTT TTGTGGGGTA CTATCAAGTA A 。

SEQ ID NO.12

1 GCTGCCACAC ATGAACATTC TGCACAGTGG TTAAACAACT ATAAAAAAGG

51 TTATGGGTAC GGACCTTACC CTCTGGGTAT AAACGGTGGA ATGCACTATG

101 GCGTTGATTT CTTCATGAAT ATTGGTACGC CAGTTAAGGC AATTTCCTCC

151 GGTAAAATCG TTGAAGCTGG TTGGTCCAAT TACGGCGGTG GTAACCAAAT

201 CGGACTTATT GAAAATGATG GGGTCCATCG TCAGTGGTAC ATGCATTTAT

251 CTAAATATAA TGTTAAGGTT GGTGATTATG TCAAAGCTGG CCAAATAATC

301 GGGTGGTCCG GAAGTACCGG TTATAGTACA GCTCCGCATT TACATTTTCA

351 AAGGATGGTT AACAGCTTCT CAAACTCCAC TGCTCAAGAT CCAATGCCCT

401 TCTTAAAGTC AGCTGGTTAT GGTAAGGCGG GCGGTACCGT TACTCCTACA

451 CCCAATACAG GTTGGAAAAC CAATAAGTAT GGAACCCTGT ACAAAAGTGA

501 GAGTGCATCT TTCACACCCA ACACCGATAT TATTACACGT ACCACCGGCC

551 CTTTTAGATC CATGCCTCAG AGTGGGGTAT TGAAAGCCGG CCAAACCATT

601 CATTATGATG AAGTCATGAA ACAAGATGGT CACGTCTGGG TTGGTTATAC

651 TGGTAATAGT GGTCAGCGTA TTTACCTACC TGTAAGAACC TGGAACAAGT

701 CTACCAATAC TTTGGGTGTG CTTTGGGGCA CCATAAAGTA A。

SEQ ID NO.13

1 GCTGCTACTC ATGAGCATTC CGCCCAATGG CTAAATAATT ACAAAAAGGG

51 TTATGGTTAT GGGCCTTATC CTCTGGGAAT CAATGGAGGT ATGCACTACG

101 GTGTAGACTT CTTCATGAAT ATAGGCACCC CGGTGAAGGC CATATCTTCA

151 GGAAAGATTG TTGAAGCGGG GTGGTCCAAT TACGGAGGTG GAAACCAAAT

201 AGGGTTAATC GAGAACGACG GTGTTCATAG GCAGTGGTAT ATGCACTTAA

251 GTAAATACAA CGTCAAGGTC GGGGATTACG TAAAGGCTGG CCAGATTATA

301 GGCTGGAGTG GAAGCACGGG GTATTCTACG GCACCCCATT TGCACTTTCA

351 ACGAATGGTC AACAGCTTTA GTAACTCGAC AGCGCAGGAC CCAATGCCGT

401 TTCTAAAATC CGCAGGTTAC GGCAAGGCGG GCGGCACCGT GACTCCAACT

451 CCGAACACAG GATGGAAAAC CAACAAATAT GGGACACTCT ACAAATCAGA

501 GTCAGCCTCG TTTACTCCCA ATACAGACAT TATTACGAGA ACCACTGGTC

551 CCTTCCGGTC TATGCCTCAA AGCGGGGTAC TTAAGGCAGG ACAGACGATT

601 CACTATGATG AAGTTATGAA ACAAGATGGC CACGTGTGGG TTGGTTATAC

651 CGGTAATTCT GGGCAGCGTA TCTACTTGCC AGTCCGCACC TGGAACAAAT

701 CTACTAATAC GCTCGGCGTG CTGTGGGGAA CAATCAAATG A。

The construction of the vector is described by taking the optimized gene sequence SEQ ID NO.3 as an example, and other gene sequences are constructed according to the same method. Amplifying a lysostaphin gene by Polymerase Chain Reaction (PCR) by using the synthetic gene as a template, adding an EcoRI restriction site (GAATTC) at the 5 'end of an upstream primer, adding a SalI restriction site (GTCGAC) at the 5' end of a downstream primer, and amplifying a lysostaphin mature gene with restriction sites at both ends, wherein the primer information is as follows:

an upstream primer: 5'-GCTGAATTCGCTGCTACTCACGAACACTC-3' (SEQ ID NO. 4),

a downstream primer: 5'-ATGGTCGACTTACTTGATAGTACCCCACA-3' (SEQ ID NO. 5),

amplifying the target gene of lysostaphin with about 0.75kb, separating the target gene by agarose gel electrophoresis, purifying and recovering the target gene according to the specification of a gene recovery kit, cutting the lysostaphin gene by using restriction enzymes EcoRI and SalI, cutting by using a culture box at 37 ℃ for 2 hours, separating the target gene by agarose gel electrophoresis, and purifying and recovering the cut lysostaphin gene according to the specification of the gene recovery kit. The digested lysostaphin gene was ligated with the pichia pastoris expression vector pPICz. Alpha.A, which was also digested with the restriction enzymes EcoRI and SalI, and T4 DNA ligase was reacted overnight at 16 ℃. The ligation product was transformed into E.coli Top10 competent cells by heat shock, plated on LB plates containing 100. Mu.g/mL bleomycin resistance, and cultured overnight at 37℃until single colonies developed. Colony PCR was performed to verify positive transformants using universal primers (5 AOX and 3 AOX) for the pPIcz. Alpha.A vector, the amplified product was verified by agarose gel electrophoresis with a target band of about 1.2kb, and the verification results are shown in FIG. 1.

5AOX：5’-GACTGGTTCCAATTGACAAGC-3’ （SEQ ID NO.6），

3AOX：5’-GCAAATGGCATTCTGACATCC-3’ （SEQ ID NO.7），

The positive transformant is sent to sequencing, and the obtained pichia pastoris expression vector pPIczalpha A-Lss-1 of mature lysostaphin is obtained. The expression of lysostaphin in the expression vector is regulated and controlled by AOX1 transcription, which is the strongest promoter of pichia pastoris, and simultaneously, the alpha-signal peptide is contained to ensure that the protein is secreted out of cells normally, and Kex2 cutting sites are contained between the alpha-signal peptide and the lysostaphin to ensure that the expressed fusion protein can be correctly cut and removed from the signal peptide. Methanol is used as an inducer to induce and express alpha-signal peptide and lysostaphin fusion protein, the fusion protein is transported to an endoplasmic reticulum under the guidance of N-terminal alpha-signal peptide, and then alpha-signal peptide is cut and removed by Kex2 protease in a Golgi body, so that natural mature lysostaphin is released, and finally secreted out of cells.

In order to facilitate separation and purification of lysostaphin, his tag can be added to lysostaphin, and the lysostaphin can be purified by Ni-NTA affinity chromatography through the His tag. The pPICz alpha A vector contains His tag at the back of the multi-cloning site, only the lysostaphin gene is needed to be inserted into the multi-cloning site when the expression vector is designed, and the termination codon (TAA) is removed, so that the expression of the protein is directly fused with the His tag on the vector, and the expression of the protein is stopped by using the termination codon (TGA) of the vector. The EcoRI and SalI cleavage sites at the multiple cloning sites were also selected as lysostaphin gene insertion sites as in the vector construction method described above. Amplifying mature lysostaphin gene with the terminator and enzyme cleavage site by using the upstream primer and His primer, carrying out double enzyme cleavage for 2h by using restriction enzymes EcoRI and SalI, then connecting with pPICz alpha A vector which is also subjected to double enzyme cleavage by using restriction enzymes EcoRI and SalI, carrying out heat shock transformation on escherichia coli, screening positive transformants, and obtaining the mature lysostaphin expression vector pPICz alpha A-LssHis containing His tag.

His primer: 5'-ATGGTCGACCTTGATAGTACCCCACAAAA-3' (SEQ ID NO. 8)

The amino acid sequence of the mature lysostaphin containing the His tag is shown as SEQ ID NO.9, and the corresponding gene sequence is shown as SEQ ID NO. 10.

SEQ ID NO.9

1 AATHEHSAQW LNNYKKGYGY GPYPLGINGG MHYGVDFFMN IGTPVKAISS

51 GKIVEAGWSN YGGGNQIGLI ENDGVHRQWY MHLSKYNVKV GDYVKAGQII

101 GWSGSTGYST APHLHFQRMV NSFSNSTAQD PMPFLKSAGY GKAGGTVTPT

151 PNTGWKTNKY GTLYKSESAS FTPNTDIITR TTGPFRSMPQ SGVLKAGQTI

201 HYDEVMKQDG HVWVGYTGNS GQRIYLPVRT WNKSTNTLGV LWGTIKVDHH

251 HHHH 。

SEQ ID NO.10

1 GCTGCTACTC ACGAACACTC TGCTCAATGG TTGAACAACT ACAAGAAGGG

51 TTACGGTTAC GGTCCATACC CATTGGGTAT CAACGGTGGT ATGCACTACG

101 GTGTTGACTT CTTCATGAAC ATCGGTACTC CAGTTAAGGC TATCTCTTCT

151 GGTAAGATCG TTGAAGCTGG TTGGTCTAAC TACGGTGGTG GTAACCAAAT

201 CGGTTTGATC GAAAACGACG GTGTTCACAG ACAATGGTAC ATGCACTTGT

251 CTAAGTACAA CGTTAAGGTT GGTGACTACG TTAAGGCTGG TCAAATCATC

301 GGTTGGTCTG GTTCTACTGG TTACTCTACT GCTCCACACT TGCACTTCCA

351 AAGAATGGTT AACTCTTTCT CTAACTCTAC TGCTCAAGAC CCAATGCCAT

401 TCTTGAAGTC TGCTGGTTAC GGTAAGGCTG GTGGTACTGT TACTCCAACT

451 CCAAACACTG GTTGGAAGAC TAACAAGTAC GGTACTTTGT ACAAGTCTGA

501 ATCTGCTTCT TTCACTCCAA ACACTGACAT CATCACTAGA ACTACTGGTC

551 CATTCAGATC TATGCCACAA TCTGGTGTTT TGAAGGCTGG TCAAACTATC

601 CACTACGACG AAGTTATGAA GCAAGACGGT CACGTTTGGG TTGGTTACAC

651 TGGTAACTCT GGTCAAAGAA TCTACTTGCC AGTTAGAACT TGGAACAAGT

701 CTACTAACAC TTTGGGTGTT TTGTGGGGTA CTATCAAGGT CGACCATCAT

751 CATCATCATC ATTGA 。

The expression vector is digested for 2 hours by the restriction enzyme PmeI, and the linearized expression vector is recovered by agarose gel electrophoresis and electrotransformed (1.5 kv-2.5 kv) to pichia pastoris competent cells, such as X33, GS115, SMD1168, KM71 and the like, preferably X33. The bacterial solution is coated on YPD plates containing bleomycin resistance and cultured for 3-5 days at 30 ℃ until single colonies are grown. And (3) selecting single bacterial colony for culture fermentation, detecting the lysostaphin enzyme activity of the fermentation supernatant, and screening to obtain the optimal expression strain.

Recombinant expression vectors with optimized gene sequences SEQ ID NO.12 and SEQ ID NO.13 are constructed by the same method, pichia pastoris cells are transformed, the lysostaphin enzyme activity of the fermentation supernatant is detected, and the optimal expression strain is obtained by screening.

EXAMPLE 2 expression and detection of recombinant lysostaphin

Shake flask culture: the expression strain of Pichia lysostaphin constructed in example 1 was selected, inoculated into 5mL of YPD medium, and cultured at 30℃and 220rpm for 24 hours to prepare a seed solution. The cells were inoculated in 50mL of BMGY medium (250 mL Erlenmeyer flask) at 1% inoculum size, cultured at 30℃for 24 hours at 220rpm, centrifuged at 4000Xg for 5 minutes, collected, resuspended in 50mL of BMMY medium, cultured at 30℃at 220rpm, induced by adding 1% methanol (final concentration) every 24 hours for 3 days, centrifuged at 4000Xg for 5 minutes, and the supernatant was collected and examined.

Fermenting in a fermentation tank: the Pichia lysostaphin expression strain constructed in example 1 was picked up, inoculated into 25mL of YPD medium, and cultured at 30℃and 220rpm for 24 hours to prepare a first seed solution. 20mL of the primary seed solution was inoculated into 200mL of BMGY medium to prepare a secondary seed solution, and the secondary seed solution was cultured at 30℃and 220rpm for 24 hours. The second-stage seed solution is fully inoculated into a 5L fermentation tank (2L BSM culture medium), the temperature is controlled to be 30+/-0.5 ℃, dissolved oxygen is controlled to be 20+/-5%, and pH=5.0+/-0.5. After basal glycerol is exhausted, 10% glycerol is fed in, after the glycerol is exhausted, until dissolved oxygen suddenly rises to 80% or more, and after half an hour of starvation, methanol is fed in to induce expression of lysostaphin, the expression is induced for 180-200 hours, and sampling is carried out at different time points for detection.

And (3) detecting antibacterial activity of lysostaphin: the antibacterial activity was evaluated using a zone of inhibition. The indicator staphylococcus aureus CMCC26003 is inoculated in LB culture medium, and cultured at 37 ℃ and 220rpm overnight to prepare indicator bacteria liquid. Diluted to OD with physiological saline ₆₂₅ =2.3, 100 μl of the dilution indicator broth was taken to 100mL of LB solid medium (temperature 50-55 ℃), mixed well, 10.5mL of solid medium was taken to a standard petri dish, cooled and solidified. Punching with a puncher, adding 5 μl of fermentation supernatant into each hole, covering with a ceramic tile cover, culturing in a 37 deg.C incubator for 16 hr, and observing the result of the inhibition zone. The detection results of the original sequence of the lysostaphin and the shake flask culture fermentation broth of the 3 optimized sequence strains are shown in fig. 2, and the results show that the 4 strains have antibacterial activity, and the antibacterial circle of the 3 optimized sequence strains is larger than that of the original sequence, which indicates that the expression quantity of the lysostaphin is obviously improved and the antibacterial activity is also improved after codon optimization. The best expression level of lysostaphin among the 3 optimized sequences is the optimized sequence1, namely SEQ ID NO.3, the GC content of the gene sequence is reduced from 52.5% to 44.4%, and the Codon Adaptability Index (CAI) is improved from 0.819 to 0.972, so that the gene is more suitable for recombinant expression of pichia pastoris. In addition, the lysostaphin liquid fused with the His purification tag also has good antibacterial activity, which is equivalent to the antibacterial activity of the lysostaphin which is not fused and expressed.

The results of the antibacterial detection of the fermentation liquor of the fermentation tank are shown in figure 2. The longer the fermentation time of the strain expressing the SEQ ID NO.3 gene sequence, the more lysostaphin is expressed, and the larger the bacteriostasis zone is formed. After 180 hours of fermentation, the fermentation broth is diluted by 10 times and still has obvious antibacterial activity. The lysostaphin expressed by pichia pastoris has good bactericidal effect on staphylococcus aureus.

Protein concentration detection: the total protein concentration of the fermentation supernatant was determined by the Bradford method. The expression curve of the lysostaphin fermented in the fermenter is shown in FIG. 3, and the highest expression level reaches 6g/L, which is the highest expression level reported at present.

Protein electrophoresis (SDS-PAGE): protein electrophoresis of lysostaphin was performed using a protein electrophoresis pre-gel from gold SpA, the results of which are shown in FIG. 4. A protein band of the target size is between the standard molecular weights of 26-34kDa, the band size being similar to the molecular weight of mature lysostaphin, about 26.9kDa. Through software analysis, the content of lysostaphin in the fermentation broth is more than 70%, the content of the hybrid protein is very small, and the separation and purification of the protein are facilitated.

Example 3 evaluation of stability

The strain stability was evaluated by serial subculture. Pichia pastoris cell colonies expressing lysostaphin constructed in example 1 were picked up and inoculated into 3mL of YPD medium, and cultured at 30℃and 220rpm for 24 hours to prepare a seed solution. 0.5mL of the seed solution was inoculated into 25mL of BMGY medium, and cultured at 30℃and 220rpm for 24 hours. The bacterial solution was transferred to a 50mL centrifuge tube, the supernatant was centrifuged (4000 Xg, room temperature), the cells were resuspended in 25mL of BMMY medium with 1% methanol, the bacterial solution was transferred to a 250mL Erlenmeyer flask and incubated at 30℃for 72 hours at 220rpm, and methanol was added to 1% concentration every 24 hours. Centrifuging (4000 Xg, room temperature) to obtain fermentation supernatant, and detecting bacteriostasis zone and protein concentration. The screened strain is selected, the strain is continuously subcultured for 10 generations, and the fermentation supernatant bacteriostasis circle is detected, so that the result shows that the bacteriostasis circle of the shake flask fermentation supernatant of each generation of strain is almost the same in size, the protein concentration is between 0.3 and 0.4g/L, and the strain stability is good.

Heat resistance stability: taking a proper amount of fermentation supernatant, heating in a water bath for 5min at 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ and 100 ℃ respectively, and observing that the fermentation broth after heat treatment is still clear, which indicates that the lysostaphin does not generate denaturation and precipitation when being heated. Centrifuging at 4000Xg for 5min, and collecting supernatant for detection of inhibition zone, wherein the result is shown in FIG. 5. The lysostaphin can endure 70 ℃, the antibacterial activity is kept unchanged, and meanwhile, the lysostaphin still has a certain antibacterial activity after being treated at 100 ℃, which shows that the lysostaphin has good thermal stability, and is particularly suitable for feed industry requiring high temperature in the feed granulating process.

EXAMPLE 4 isolation and purification of recombinant lysostaphin

The fermentation supernatant in example 2 was collected and desalted using a roll film, and water was added multiple times until the ionic strength of the desalted liquid was less than 13ms/cm. The desalted solution was filtered with a 0.45 μm filter membrane, and the filtered desalted solution was collected. The CM-Sepharose Fast Flow column was equilibrated with 10 bed volumes of binding buffer at a flow rate of 1 bed volume/min. The filtered desalted solution was passed to a column at a flow rate of 1 bed volume/min. The column was flushed with 5 bed volumes of binding buffer. The column was rinsed with 5 bed volumes of 0.3M eluent to elute the impurity proteins at a flow rate of 1 bed volume/min. The column was washed with 5 bed volumes of 0.5M eluent, eluting the protein of interest at a flow rate of 1 bed volume/min. Detecting protein absorbance of the eluent by a nucleic acid protein detector, and collecting the eluent with protein absorption peaks. The eluent is lysostaphin eluent. Concentrating and desalting by using a roll membrane until the ionic strength of the eluent is less than 1ms/cm, and obtaining the recombinant lysostaphin purified sample. As shown in FIG. 4, the result of protein electrophoresis of the purified protein showed that there was only a single band of interest and almost no foreign proteins. The Pichia pastoris is fermented by adopting an inorganic salt culture medium, so that protein impurities are few, and the Pichia pastoris secretes few hetero proteins, so that the obtained fermentation supernatant has high lysostaphin content and few hetero proteins, and a purified protein sample with high purity can be obtained by only one-step ion exchange.

Binding buffer: 3.8mM NaH ₂ PO ₄ ，16.2mM Na ₂ HPO ₄ 50mM NaCl.0.3M eluent: 3.8mM NaH ₂ PO ₄ ，16.2mM Na ₂ HPO ₄ 0.3M NaCl.0.5M eluent: 3.8mM NaH ₂ PO ₄ ，16.2mM Na ₂ HPO ₄ 0.5M NaCl. The above-mentioned eluent was dissolved and filtered through a 0.22 μm filter membrane.

If the His-tag-containing lysostaphin fusion protein is expressed, the lysostaphin can be recovered by specific purification using a Ni-NTA affinity chromatography method. The Ni-NTA column was equilibrated with 10 bed volumes of equilibration buffer at a flow rate of 1 bed volume/min. The fermentation supernatant can be directly put on a column for protein purification, and the flow rate is 1 time of the volume of a bed per minute. 5 times of the column bed volume of the balance buffer solution washes the chromatographic column, 5 times of the column bed volume of the combination buffer solution washes the chromatographic column, and impurities are eluted. The column was washed with 5 bed volumes of 100-500mM eluent, eluting the protein of interest at a flow rate of 1 bed volume/min. Detecting protein absorbance of the eluent by a nucleic acid protein detector, and collecting the eluent with protein absorption peaks. The eluent is lysostaphin eluent. Concentrating and desalting by using a roll membrane until the ionic strength of the eluent is less than 1ms/cm, and obtaining the recombinant lysostaphin purified sample.

Equilibration buffer: 9.5mM NaH ₂ PO ₄ ，40.5mM Na ₂ HPO ₄ 500mM NaCl, pH7.4. Binding buffer: 9.5mM NaH ₂ PO ₄ ，40.5mM Na ₂ HPO ₄ 500mM NaCl,10mM imidazole, pH7.4. Eluent: 9.5mM NaH ₂ PO ₄ ，40.5mM Na ₂ HPO ₄ 500mM NaCl,100-500mM imidazole, pH7.4. The solution was dissolved and filtered through a 0.22 μm filter membrane.

Sequence listing

<110> Guangdong Yiduoli Biotech stock Co., ltd

<120> a method for efficiently expressing a lysostaphin

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 493

<212> PRT

<213> Staphylococcus simulans

<400> 1

Met Lys Lys Thr Lys Asn Asn Tyr Tyr Thr Arg Pro Leu Ala Ile Gly

1 5 10 15

Leu Ser Thr Phe Ala Leu Ala Ser Ile Val Tyr Gly Gly Ile Gln Asn

20 25 30

Glu Thr His Ala Ser Glu Lys Ser Asn Met Asp Val Ser Lys Lys Val

35 40 45

Ala Glu Val Glu Thr Ser Lys Ala Pro Val Glu Asn Thr Ala Glu Val

50 55 60

Glu Thr Ser Lys Ala Pro Val Glu Asn Thr Ala Glu Val Glu Thr Ser

65 70 75 80

Lys Ala Pro Val Glu Asn Thr Ala Glu Val Glu Thr Ser Lys Ala Pro

85 90 95

Val Glu Asn Thr Ala Glu Val Glu Thr Ser Lys Ala Pro Val Glu Asn

100 105 110

Thr Ala Glu Val Glu Thr Ser Lys Ala Pro Val Glu Asn Thr Ala Glu

115 120 125

Val Glu Thr Ser Lys Ala Pro Val Glu Asn Thr Ala Glu Val Glu Thr

130 135 140

Ser Lys Ala Pro Val Glu Asn Thr Ala Glu Val Glu Thr Ser Lys Ala

145 150 155 160

Pro Val Glu Asn Thr Ala Glu Val Glu Thr Ser Lys Ala Pro Val Glu

165 170 175

Asn Thr Ala Glu Val Glu Thr Ser Lys Ala Pro Val Glu Asn Thr Ala

180 185 190

Glu Val Glu Thr Ser Lys Ala Pro Val Glu Asn Thr Ala Glu Val Glu

195 200 205

Thr Ser Lys Ala Pro Val Glu Asn Thr Ala Glu Val Glu Thr Ser Lys

210 215 220

Ala Pro Val Glu Asn Thr Ala Glu Val Glu Thr Ser Lys Ala Leu Val

225 230 235 240

Gln Asn Arg Thr Ala Leu Arg Ala Ala Thr His Glu His Ser Ala Gln

245 250 255

Trp Leu Asn Asn Tyr Lys Lys Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu

260 265 270

Gly Ile Asn Gly Gly Met His Tyr Gly Val Asp Phe Phe Met Asn Ile

275 280 285

Gly Thr Pro Val Lys Ala Ile Ser Ser Gly Lys Ile Val Glu Ala Gly

290 295 300

Trp Ser Asn Tyr Gly Gly Gly Asn Gln Ile Gly Leu Ile Glu Asn Asp

305 310 315 320

Gly Val His Arg Gln Trp Tyr Met His Leu Ser Lys Tyr Asn Val Lys

325 330 335

Val Gly Asp Tyr Val Lys Ala Gly Gln Ile Ile Gly Trp Ser Gly Ser

340 345 350

Thr Gly Tyr Ser Thr Ala Pro His Leu His Phe Gln Arg Met Val Asn

355 360 365

Ser Phe Ser Asn Ser Thr Ala Gln Asp Pro Met Pro Phe Leu Lys Ser

370 375 380

Ala Gly Tyr Gly Lys Ala Gly Gly Thr Val Thr Pro Thr Pro Asn Thr

385 390 395 400

Gly Trp Lys Thr Asn Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala

405 410 415

Ser Phe Thr Pro Asn Thr Asp Ile Ile Thr Arg Thr Thr Gly Pro Phe

420 425 430

Arg Ser Met Pro Gln Ser Gly Val Leu Lys Ala Gly Gln Thr Ile His

435 440 445

Tyr Asp Glu Val Met Lys Gln Asp Gly His Val Trp Val Gly Tyr Thr

450 455 460

Gly Asn Ser Gly Gln Arg Ile Tyr Leu Pro Val Arg Thr Trp Asn Lys

465 470 475 480

Ser Thr Asn Thr Leu Gly Val Leu Trp Gly Thr Ile Lys

485 490

<210> 2

<211> 246

<212> PRT

<213> Staphylococcus simulans (mimicking Staphylococcus)

<400> 2

Ala Ala Thr His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys Lys

1 5 10 15

Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met His

20 25 30

Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala Ile

35 40 45

Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly Gly

50 55 60

Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp Tyr

65 70 75 80

Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys Ala

85 90 95

Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala Pro

100 105 110

His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr Ala

115 120 125

Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala Gly

130 135 140

Gly Thr Val Thr Pro Thr Pro Asn Thr Gly Trp Lys Thr Asn Lys Tyr

145 150 155 160

Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Pro Asn Thr Asp

165 170 175

Ile Ile Thr Arg Thr Thr Gly Pro Phe Arg Ser Met Pro Gln Ser Gly

180 185 190

Val Leu Lys Ala Gly Gln Thr Ile His Tyr Asp Glu Val Met Lys Gln

195 200 205

Asp Gly His Val Trp Val Gly Tyr Thr Gly Asn Ser Gly Gln Arg Ile

210 215 220

Tyr Leu Pro Val Arg Thr Trp Asn Lys Ser Thr Asn Thr Leu Gly Val

225 230 235 240

Leu Trp Gly Thr Ile Lys

245

<210> 3

<211> 741

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gctgctactc acgaacactc tgctcaatgg ttgaacaact acaagaaggg ttacggttac 60

ggtccatacc cattgggtat caacggtggt atgcactacg gtgttgactt cttcatgaac 120

atcggtactc cagttaaggc tatctcttct ggtaagatcg ttgaagctgg ttggtctaac 180

tacggtggtg gtaaccaaat cggtttgatc gaaaacgacg gtgttcacag acaatggtac 240

atgcacttgt ctaagtacaa cgttaaggtt ggtgactacg ttaaggctgg tcaaatcatc 300

ggttggtctg gttctactgg ttactctact gctccacact tgcacttcca aagaatggtt 360

aactctttct ctaactctac tgctcaagac ccaatgccat tcttgaagtc tgctggttac 420

ggtaaggctg gtggtactgt tactccaact ccaaacactg gttggaagac taacaagtac 480

ggtactttgt acaagtctga atctgcttct ttcactccaa acactgacat catcactaga 540

actactggtc cattcagatc tatgccacaa tctggtgttt tgaaggctgg tcaaactatc 600

cactacgacg aagttatgaa gcaagacggt cacgtttggg ttggttacac tggtaactct 660

ggtcaaagaa tctacttgcc agttagaact tggaacaagt ctactaacac tttgggtgtt 720

ttgtggggta ctatcaagta a 741

<210> 4

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gctgaattcg ctgctactca cgaacactc 29

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atggtcgact tacttgatag taccccaca 29

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gactggttcc aattgacaag c 21

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gcaaatggca ttctgacatc c 21

<210> 8

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atggtcgacc ttgatagtac cccacaaaa 29

<210> 9

<211> 254

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Ala Ala Thr His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys Lys

1 5 10 15

Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met His

20 25 30

Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala Ile

35 40 45

Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly Gly

50 55 60

Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp Tyr

65 70 75 80

Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys Ala

85 90 95

Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala Pro

100 105 110

His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr Ala

115 120 125

Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala Gly

130 135 140

Gly Thr Val Thr Pro Thr Pro Asn Thr Gly Trp Lys Thr Asn Lys Tyr

145 150 155 160

Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Pro Asn Thr Asp

165 170 175

Ile Ile Thr Arg Thr Thr Gly Pro Phe Arg Ser Met Pro Gln Ser Gly

180 185 190

Val Leu Lys Ala Gly Gln Thr Ile His Tyr Asp Glu Val Met Lys Gln

195 200 205

Asp Gly His Val Trp Val Gly Tyr Thr Gly Asn Ser Gly Gln Arg Ile

210 215 220

Tyr Leu Pro Val Arg Thr Trp Asn Lys Ser Thr Asn Thr Leu Gly Val

225 230 235 240

Leu Trp Gly Thr Ile Lys Val Asp His His His His His His

245 250

<210> 10

<211> 765

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gctgctactc acgaacactc tgctcaatgg ttgaacaact acaagaaggg ttacggttac 60

ggtccatacc cattgggtat caacggtggt atgcactacg gtgttgactt cttcatgaac 120

atcggtactc cagttaaggc tatctcttct ggtaagatcg ttgaagctgg ttggtctaac 180

tacggtggtg gtaaccaaat cggtttgatc gaaaacgacg gtgttcacag acaatggtac 240

atgcacttgt ctaagtacaa cgttaaggtt ggtgactacg ttaaggctgg tcaaatcatc 300

ggttggtctg gttctactgg ttactctact gctccacact tgcacttcca aagaatggtt 360

aactctttct ctaactctac tgctcaagac ccaatgccat tcttgaagtc tgctggttac 420

ggtaaggctg gtggtactgt tactccaact ccaaacactg gttggaagac taacaagtac 480

ggtactttgt acaagtctga atctgcttct ttcactccaa acactgacat catcactaga 540

actactggtc cattcagatc tatgccacaa tctggtgttt tgaaggctgg tcaaactatc 600

cactacgacg aagttatgaa gcaagacggt cacgtttggg ttggttacac tggtaactct 660

ggtcaaagaa tctacttgcc agttagaact tggaacaagt ctactaacac tttgggtgtt 720

ttgtggggta ctatcaaggt cgaccatcat catcatcatc attga 765

<210> 11

<211> 741

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gctgctaccc acgagcactc cgctcaatgg ttgaacaact acaagaaggg ttacggttac 60

ggtccatacc cattgggtat caacggtggt atgcactacg gtgttgactt cttcatgaac 120

atcggtaccc cagtcaaggc tatctcctcc ggtaagatcg tcgaggctgg ttggtccaac 180

tacggtggtg gtaaccaaat cggtttgatc gagaacgacg gtgtccacag acaatggtac 240

atgcacttgt ccaagtacaa cgtcaaggtc ggtgactacg tcaaggctgg tcaaatcatc 300

ggttggtccg gttccaccgg ttactccacc gctccacact tgcacttcca aagaatggtc 360

aactccttct ccaactccac cgctcaagac ccaatgccat tcttgaagtc cgctggttac 420

ggtaaggctg gtggtaccgt caccccaacc ccaaacaccg gttggaagac caacaagtac 480

ggtaccttgt acaagtccga gtccgcttcc ttcaccccaa acaccgacat catcaccaga 540

accaccggtc cattcagatc catgccacaa tccggtgtct tgaaggctgg tcaaaccatc 600

cactacgacg aggtcatgaa gcaagacggt cacgtctggg tcggttacac cggtaactcc 660

ggtcaaagaa tctacttgcc agtcagaacc tggaacaagt ccaccaacac cttgggtgtc 720

ttgtggggta ccatcaagta a 741

<210> 12

<211> 741

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gctgccacac atgaacattc tgcacagtgg ttaaacaact ataaaaaagg ttatgggtac 60

ggaccttacc ctctgggtat aaacggtgga atgcactatg gcgttgattt cttcatgaat 120

attggtacgc cagttaaggc aatttcctcc ggtaaaatcg ttgaagctgg ttggtccaat 180

tacggcggtg gtaaccaaat cggacttatt gaaaatgatg gggtccatcg tcagtggtac 240

atgcatttat ctaaatataa tgttaaggtt ggtgattatg tcaaagctgg ccaaataatc 300

gggtggtccg gaagtaccgg ttatagtaca gctccgcatt tacattttca aaggatggtt 360

aacagcttct caaactccac tgctcaagat ccaatgccct tcttaaagtc agctggttat 420

ggtaaggcgg gcggtaccgt tactcctaca cccaatacag gttggaaaac caataagtat 480

ggaaccctgt acaaaagtga gagtgcatct ttcacaccca acaccgatat tattacacgt 540

accaccggcc cttttagatc catgcctcag agtggggtat tgaaagccgg ccaaaccatt 600

cattatgatg aagtcatgaa acaagatggt cacgtctggg ttggttatac tggtaatagt 660

ggtcagcgta tttacctacc tgtaagaacc tggaacaagt ctaccaatac tttgggtgtg 720

ctttggggca ccataaagta a 741

<210> 13

<211> 741

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

gctgctactc atgagcattc cgcccaatgg ctaaataatt acaaaaaggg ttatggttat 60

gggccttatc ctctgggaat caatggaggt atgcactacg gtgtagactt cttcatgaat 120

ataggcaccc cggtgaaggc catatcttca ggaaagattg ttgaagcggg gtggtccaat 180

tacggaggtg gaaaccaaat agggttaatc gagaacgacg gtgttcatag gcagtggtat 240

atgcacttaa gtaaatacaa cgtcaaggtc ggggattacg taaaggctgg ccagattata 300

ggctggagtg gaagcacggg gtattctacg gcaccccatt tgcactttca acgaatggtc 360

aacagcttta gtaactcgac agcgcaggac ccaatgccgt ttctaaaatc cgcaggttac 420

ggcaaggcgg gcggcaccgt gactccaact ccgaacacag gatggaaaac caacaaatat 480

gggacactct acaaatcaga gtcagcctcg tttactccca atacagacat tattacgaga 540

accactggtc ccttccggtc tatgcctcaa agcggggtac ttaaggcagg acagacgatt 600

cactatgatg aagttatgaa acaagatggc cacgtgtggg ttggttatac cggtaattct 660

gggcagcgta tctacttgcc agtccgcacc tggaacaaat ctactaatac gctcggcgtg 720

ctgtggggaa caatcaaatg a 741

Claims

1. A method for efficiently expressing lysostaphin is characterized by comprising the steps ofPichia pastoris) The nucleotide sequence of the optimized lysostaphin encoding gene is shown as SEQ ID NO.3, SEQ ID NO.12 or SEQ ID NO. 13.

2. A method of biologically producing lysostaphin, said method comprising the steps of:

in Pichia pastorisPichia pastoris) The optimized lysostaphin encoding gene is expressed in the medium, wherein the nucleotide sequence of the optimized lysostaphin encoding gene is shown as SEQ ID NO.3, SEQ ID NO.12 or SEQ ID NO. 13;

separating lysostaphin in the fermentation supernatant.

3. The method for biologically preparing lysostaphin as claimed in claim 2, wherein the lysostaphin in the fermentation supernatant is isolated and purified by one-step ion exchange or affinity chromatography.