Soluble expression and purification of a crab antimicrobial peptide scygonadin in different expression plasmids and analysis of its antimicrobial activity.
Scygonadin is an anionic antimicrobial peptide recently identified from the seminal plasma of Scylla serrata. To gain more detailed information on its antimicrobial activity, scygonadin mature peptide was expressed in Escherichia coli in order to obtain a large quantity of biologically active product. An approximately 43 kDa fusion protein CKS-scygonadin was obtained in a highly stable and soluble form. The soluble component of the fusion CKS-scygonadin was purified by immobilized metal affinity chromatography (IMAC). A single 11 kDa recombinant scygonadin was cleaved from CKS-scygonadin and purified from the cleavage mixture using an affinity chromatography column with a yield of 10.6 mg/L. Alternatively, a recombinant scygonadin was purified from pET28-scygonadin by one-step Ni(2+) affinity chromatography and 65.9 mg/L pure recombinant scygonadin was obtained which was higher than that purified from pTrc-CKS/scygonadin in bacteria culture. The recombinant scygonadin was confirmed using SDS-PAGE analysis and MS-fingerprinting. Both recombinant products of scygonadin from different expressed plasmids showed the activity against both Gram-positive and Gram-negative bacteria, but no activity against yeast and fungi tested. The kinetic studies showed that the recombinant scygonadin was strong active against Staphylococcus aureus and the killing of S. aureus appeared time and dose dependent. Considering the quantity of recombinant product and the applicability of purification, the pET28-scygonadin expression system is a better choice to produce large quantities of recombinant scygonadin for commercial use in future. This is the first report on the heterologous expression of antimicrobial peptide scygonadin in E. coli.
Peng
Hui
H
State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen, Fujian 361005, PR China.
Yang
Ming
M
Huang
Wen-Shu
WS
Ding
Jian
J
Qu
Hai-Dong
HD
Cai
Jing-Jing
JJ
Zhang
Nai
N
Wang
Ke-Jian
KJ
D000818:Animals; D000890:Anti-Infective Agents / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003386:Brachyura / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D011994:Recombinant Proteins