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Optimized Expression of Antimicrobial Peptide BhSGAMP-1-S and Analysis on Its Antimicrobial Activity Analysis

Author: WangZuo
Tutor: WangDun
School: Northwest University of Science and Technology
Course: Forest Protection
Keywords: antimicrobial peptides gene modification BhSGAMP-1-S soluble expression antimicrobial assay
CLC: Q786
Type: Master's thesis
Year: 2010
Downloads: 102
Quote: 1
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Abstract


Antimicrobial peptides (AMPs) are one of the most important effector molecules of the innate immune system, which are evolutionarily conserved component of the principal defence system for the majority of living organisms, and are found among all classes of life including insects, amphibians and mammals. AMPs not only have broad-spectrum antibacterial activity, some of them even have the activities against viruses, fungi and tumour cell, but they are harmless to normal cells tisues of host. Further more, They have the characteristics of small molecular weight, fast synthesis, rapid diffusability, strong alkalinity, thermo stability, and good solubility. Therefore, AMPs have received increasing attentions as potential new antimicrobial substances for use in therapeutics, animal drugs, and food preservatives.BhSGAMP-1 is one of the AMPs involved in a preventive mechanism of defense of the fly Bradysia hygida which is expressed exclusively in the salivary glands of the larvae while they are preparing to ecdysis. Secretion of BhSGAMP-1 in the saliva could help prevent microbial infection during ecdysis, by killing harmful microorganisms in the immediate vicinity of the animal. The nature BhSGAMP-1 has broad-spectrum antibacterial activity and there is no report about pruducing the BhSGAMP-1-S by genetic engineering. In this study, we report a method to express it in Escherichia coli and purify it and analyzed the antimicrobial activities of the purified recombinant BhSGAMP-1-S by bioassay for the first time.The BhSGAMP-1-S gene based on the reported cDNA sequence of Bradysia hygida salivary gland antimicrobial peptide 1 in Genbank was synthesized with preferred codons of E.coli. It was expressed as a fusion protein in E.coli TB1 by using pMAL-c2X as vector. We designed tests with avariety induced temperature and time conditions, in order to improve the amount of the product and obtain the best induced parameters. The fusion protein was mostly expressed in soluble form with a theoretical molecular weight of 50 kDa under the optimized conditions at high level (more than 52.6% of the total proteins). After purified by Amylase Resin affinity chromatography, the fusion protein was cleaved by enterokinase to release recombinant BhSGAMP-1-S. Recombinant BhSGAMP-1-S with molecular weight of 7.2 kDa was purified to homogeneity by RP-HPLC after removing the contaminants by molecular exclusion chromatography and 0.38 mg of pure BhSGAMP-1-S was obtained from 100 mL culture medium. Antimicrobial assays demonstrated that the recombinant BhSGAMP-1-S expressed in E. coli was active against several Gram-positive, Gram-negative bacteria and fungus.In this study, we constructed the engineered bacteria with optimized BhSGAMP-1-S gene and got the high purity recombinant BhSGAMP-1-S. We provided a useful approach to the production of the recombinant antimicrobial peptide with high biological activity and established a good foundation for further study and use of this antimicrobial peptide.

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