Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27.
This study reports the first successful recombinant expression of cationic antimicrobial peptides human beta-defensin-26 and human beta-defensin-27 in Escherichia coli. HBD26 and HBD27 genes were synthesized through codon optimization, and each gene was then cloned into the expression vector pET32, which feature fusion protein thioredoxin at the N-terminal. The recombinant plasmids were then transformed into E. coli BL21 (DE3) and cultured in MBL medium, which gave yields of HBD26 and HBD27 fusion proteins of up to 1.38 and 1.29 g l(-1), respectively. Affinity chromatography was used to purify the soluble fusion proteins, and the N-terminal TrxA tags were cleaved off by enterokinase. Pure HBD26 and HBD27 were then obtained by cationic exchange chromatography. The overall recovery of HBD26 was 38% and that of HBD27 reached 36%. Both variants showed salt-sensitive antimicrobial activity against gram-negative E. coli but not against gram-positive Staphylococcus aureus and Saccharomyces cerevisiae.
Huang
Lei
L
School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore, 637459, Singapore.
Leong
Susanna Su Jan
SS
Jiang
Rongrong
R
D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000494:pharmacology; D001483:Base Sequence; D002846:Chromatography, Affinity; D015169:Colony Count, Microbial; D004765:Enteropeptidase / Q000378:metabolism; D030501:Epididymal Secretory Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D013879:Thioredoxins / Q000302:isolation & purification; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
D000900:Anti-Bacterial Agents; C426184:DEFB126 protein, human; D030501:Epididymal Secretory Proteins; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins; C500653:beta-defensin-27, human; D013879:Thioredoxins; D004765:Enteropeptidase