High-level expression and novel purification strategy of recombinant thanatin analog in Escherichia coli.
Recombinant thanatin analog (TH1) is a cationic 20-amino-acid antibacterial peptide with a conserved cysteine disulfide bond. It exhibits a broad antibacterial spectrum. Different strategies have been developed to produce small antibacterial peptides using recombinant techniques. To date, no efforts to obtain large quantities of active recombinant TH1 have been reported. This study describes the synthesis of TH1 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released TH1. By constructing the expression plasmid (pET32a-TH1), high yields of soluble TH1 fusion protein (0.416 g/L) can be obtained in E. coli. Further optimization studies have been carried out to increase the expression of TH1 in different culture conditions, with the final amount of pure TH1 being 13.2 mg/L. The results show that the expression system provides a simple and reliable strategy for generating large quantities of TH1 by soluble fusion expression in E. coli.
Wu
G-Q
GQ
Clinical Hospital, Southeast University, Nanjing 210009, PRC.
Li
L-X
LX
Ding
J-X
JX
Wen
L-Z
LZ
Shen
Z-L
ZL
D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008826:Microbial Sensitivity Tests; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D023181:Antimicrobial Cationic Peptides; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; C098076:thanatin