Effect of fusion expression of HLH domain on antibacterial activity of Sus scrofa lysozyme
[Objective]To improve the anti-Gram-negative of Sus scrofa lysozyme(SSL)by genetic manipulation.[Meth-od]A helix-loop-helix(HLH)domain containing a functional peptide was obtained by molecular simulation of SSL.Then,the HLH coding gene was fused with the N-terminal or c-terminal of SSL gene,the recombinant proteins were expressed in E.coli,and their antibacterial activity was detected after renaturation and purification.At last,the bactericidal mechanism of the fusion protein with high antibacterial activity was investigated by atomic force microscopy and fluorescence staining.[Result]Compared with the control group,the N-terminal and c-terminal fusion proteins maintained the activity of SSL against Gram-positive bacteria,and the antibacterial activity of both fusion proteins against Gram-negative was significantly enhanced,the activity ofN-terminal fusion product was higher,the antibacterial coefficients against E.coli ATCC 10798,E.coli ATCC 25922,Klebsiella TR5,Pseudomonas aeruginosa ATCC 15442 and Salmonella CMCC(B)50335 were 1.64,1.24,2.56,1.72 and 1.42 respectively;and the MIC were 90 μg/mL,100 μg/mL,40 μg/mL,80 μg/mL and 100 μg/mL,respectively.The fu-sion protein was found to significantly enhance the permeability of the Gram-negative cell membrane.[Conclusion]The anti-Gram-negative of SSL was significantly enhanced by fusion expression of HLH domain,which may provide reference for the activities improvement of other lysozymes.
Sus scrofa lysozymeantimicrobial activityantibacterial peptideHelix-loop-helix(HLH)motifmolecular sim-ulationfusion expressionbactericidal mechanismpenetrability
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