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| Expression of Human Lysozyme-like Protein 6 in Pichia pastoris and Analysis of Enzymatic Activity of the Protein |
| HUANG Peng1, LI Wen-shu3, XIE Jun4, BAO Jian-ying1, CAO Xiao-e1, YU Long4, XU Yi-xin2 |
1. College of Basic Medicine, Shanghai University of Medicine and Health Sciences, Shanghai 200237, China;
2. College of Pharmacy, Shanghai University of Medicine and Health Sciences, Shanghai 200237, China;
3. College of Arts and Sciences, New York University, Shanghai 200122, China;
4. State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433, China |
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Abstract The aim is to realize the recombinant expression of human lysozyme-like protein 6 (hLyzl6) by the Pichia pastoris expression system and to investigate its enzymatic activity. The hLyzl6 gene was synthesized chemically according to the codon usage preference of Pichia pastoris and cloned into the expression vector pPIC9K. The recombinant plasmid was linearized and transformed into competent Pichia pastoris GS115. After screening by G418 plate, the yeast strain was cultured and induced with methanol. The recombinant protein was purified by chitin affinity chromatography and subjected to activity assay. The results showed that the lytic activity in the culture supernatant reached the peak after 72 h of induction and the existence of the recombinant protein was confirmed by SDS-PAGE. The recombinant hLyzl6 exhibited bactericidal activity against Micrococcus lysodeikticus by using turbidimetric assay and had the activity of 54 700 U/mg. The optimum temperature and pH for hLyzl6 were 40℃ and 5.5 respectively and Cu2+ was found to have strong inhibition effect on hLyzl6. The EC50 value was 30.2799 mg/L. In conclusion, recombinant hLyzl6 was expressed successfully by the Pichia pastoris strain GS115 and had in vitro bactericidal activity, which indicated that hLyzl6 may play a role in innate immunity of the male reproductive system and laid a foundation for its functional research and application.
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Received: 10 July 2015
Published: 25 August 2015
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