Heterologous Expression of Cyclina sinensis Mytimacin Antibacterial Peptide Based on Recombinant Pichia pastoris

doi:10.13560/j.cnki.biotech.bull.1985.2019-0700

Abstract

Abstract: Mytimacin is a member of the Macin antimicrobial peptide family expressed in invertebrates. It is a favorable candidate to develop natural antimicrobial agents by recombinant DNA technology because of its strong antimicrobial activity against pathogens. RT-PCR was used to clone the gene encoding mytimacin mature peptide from the adductor muscle of Cyclina sinensis;the Xho I restriction site and the signal peptidase recognition site were added to its 5' end,and the Xba I restriction site and a 6×His-tag to its 3' end after three times of PCRs the acquired target gene was named “CsMm”. The recombinant Pichia pastoris X-33/pPICZαA-CsMm was constructed using pPICZαA as an expression vector and P. pastoris X-33 as an engineering strain. The yeast transformants containing multicopy gene insertions screened by high-concentration zeocin were induced for 72 h with 1.5% methanol at 28℃ and 250 r/min,and the acquired culture medium supernatant was purified by immobilized metal affinity chromatography（IMAC）;the purified protein was identified by MALDI-TOF-TOF mass spectrometry analysis. In addition,the antibacterial activity of the recombinant CsMm was detected by coating method and turbidimetric method. The results showed that the heterologous expression based on the recombinant P. pastoris X-33/pPICZαA-CsMm a recombinant protein with an expression level of 25.6 mg/L;MALDI-TOF-TOF mass spectrometry analysis demonstrated that the purified recombinant protein was the expected recombinant CsMm with a molecular weight of 7.8 kD. The bacteriostasis test showed that the recombinant CsMm had obvious antibacterial activity against Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Vibrio Parahemolyticus. The recombinant P. pastoris X-33/pPICZαA-CsMm constructed in the present study can effectively synthesize the bioactive recombinant C. sinensis Mytimacin,which provides a technical approach for the development of natural small molecule antibacterial agent from shellfish.

Key words: Cyclina sinensis, Mytimacin, Pichia pastoris, recombinant expression, antibacterial activity

ZHAO Zhen, WANG Sha-sha, LÜ Xing-xing, TAO Yan, XIE Jing, QIAN Yun-fang. Heterologous Expression of Cyclina sinensis Mytimacin Antibacterial Peptide Based on Recombinant Pichia pastoris[J]. Biotechnology Bulletin, 2020, 36(5): 150-158.

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