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Recombinant Expression and Biological Activities of the Antimicrobial Bomains Derived from Granulysin

Author: LiangLin
Tutor: ChaXiangDong
School: Anhui University
Course: Cell Biology
Keywords: Gl3 domain SOS reponse Saccharomyces cerevisiae factor secretory expression
CLC: Q51
Type: Master's thesis
Year: 2011
Downloads: 5
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Abstract


G13 fragment of granulysin is an amphiphilic cationic antimicrobial peptide which has a-helical structures. In this study, we used PCR to amplify a-factor from the expression vector pPICZaA of Saccharomyces cerevisiae, and transformed a-factor to change the incomplete cutting conditions. The transformed a-factor was cloned into the pYES2 plasmid, then the pYES2-a expression and secretion vector was constructed. Inserted G13 domain into the pYES2-a vector to construct pYES2-a-G13 plasmid, and induced in Saccharomyces cerevisiae INVScl, then detected by Tricine-SDS-PAGE electrophoresis. The experimental results suggested that the recombinant G13 domain the expression domain was directly secreted into the extracellular, and preliminary identificated that G13 domain had antimicrobial activity for E.coli DH5a. This study will step to simplify separation and purification and lay the foundation for antimicrobial activity in the further.In addition, by using self-luminous lux gene as a reporter gene we detected the effects of G13 domain of granulysin on the expression of the reporter gene under the control of the SOS promoter. Our results showed that recombinant G13 domain did induce the expression of the reporter gene, and the expression level was time-dependent and took on the form of a peak curve. At about 4 min of inducton, the expression level of the reporter gene reached its peak, and then gradually decreased. The experimental results suggested that the recombinant G13 domain triggered the degradation of the repressor protein LexA, and induced SOS response in the bacterial cells.Furthermore, we preliminary studied electroeluting method for purification of NKLF-2 domain which had 67% sequence similarity with the experiment of the G13 domain structure. After pACYC-NKLF2 BL21 bacteria was inducted, NKLF-2 was separated from other protein by Tricine-SDS-PAGE electrophoresis. We cutted the gels with the target protein, and got NKLF-2 from the gel into dialysis bags by electrophoresis. NKLF-2 was dialysised with PBS buffer and precipitated with acetone, than detected by Tricine-SDS-PAGE Electrophoresis. The experimental results suggested that recombinant NKLF-2 domain was purified by electroeluting method, and provided another technical method for study of structure and function by mass spectrometry and circular dichroism in the further.

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CLC: > Biological Sciences > Biochemistry > Protein
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