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| Modification of 5’UTR Sequences of pPIC9K Increases Expression of Antimicrobial Peptide PR39 |
| MING Fei-ping1,2, YANG Jun1, ZHU Jin-mei1, KUANG Zhe-shi3, LI Hua-zhou4, XIA Feng-geng2, YE Ming-qiang3, WANG Hou-guang4, ZHAO Xiang-jie3, HUANG Zhi-feng1, MA Miao-peng1, SHI Ju-qing1, CAI Hai-ming1, ZHANG Ling-hua1 |
1. College of Life Science, South China Agricultural University, Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, Guangzhou 510642, China;
2. Guangzhou Institute of Microbiology, Guangzhou 510663, China;
3. Bio-Tech Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
4. Guangzhou Fine Breed Swine Farm, Guangzhou 510540, China |
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Abstract Objective: To express antibacterial peptide PR39 in Pichia pastoris and determine the activity of product.Methods: Four oligonucleotide fragments were synthesized according to the codon bias of Pichia pastoris,and complete coding sequence was obtained by overlapping PCR, then the sequence was cloned to expression vector pPIC9K. The sequence by Kex2 to the first nucleotide of PR39 was knocked out on the recombinant plasmid pPIC9K-PR39, then the sequence of GGATCCAA in 5'UTR was also deleted by PCR-Restriction Enzyme ligation method, finally a new expression vector named pPIC9K-PR39-D-E was obtained. The vector pPIC9K-PR39-D-E was transformed to Pichia pastoris SMD1168, and clones were identified by PCR and than screened with G418 for expression under induction of 0.5% methanol. The expressed product was identified by Tricine-SDS-PAGE, and determined for antibacterial activity by agarose diffusion test,purified by reversed phase chromatography and ion-exchange column chromatography. Results: PR39 with 4.7kDa and reached 175.6mg/L after purification. It showed antibacterial activity to E.coli DH5α. Conclusion: PR39 was successfully expressed in Pichia pastoris, which laid a foundation of further study on antibacterial peptide.
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Received: 19 August 2013
Published: 25 December 2013
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Cite this article:
MING Fei-ping, YANG Jun, ZHU Jin-mei, KUANG Zhe-shi, LI Hua-zhou, XIA Feng-geng, YE Ming-qiang, WANG Hou-guang, ZHAO Xiang-jie, HUANG Zhi-feng, MA Miao-peng, SHI Ju-qing, CAI Hai-ming, ZHANG Ling-hua. Modification of 5’UTR Sequences of pPIC9K Increases Expression of Antimicrobial Peptide PR39. China Biotechnology, 2013, 33(12): 86-91.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2013/V33/I12/86
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