High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression.
Human beta-defensin-4 (hBD4) is a cationic 50-amino acid antimicrobial peptide with three conserved cysteine disulfide bonds. It exhibits a broad antimicrobial spectrum. This study describes the synthesis of hBD4 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released hBD4. A PCR-based gene SOEing (splicing by overlap extension) synthesis method was used in the synthesis of the hBD4 gene with optimized codons. By constructing the expression plasmid (pET32-smhBD4), high concentration of soluble hBD4 fusion protein (1.9 g/l) can be obtained in E. coli. Further optimization studies showed that the expression system was very efficient to produce soluble target protein, and the solubility of the target protein could attain more than 99% even when the culture temperature was as high as 37 degrees C. The highest productivity (2.68 g/l) of the hBD4 fusion protein was achieved by cultivating the E. coli (pET32-smhBD4) in MBL medium at 34 degrees C, inducing the culture at the mid-exponential phase with 0.4-mM isopropyl beta-D-galactopyranoside (IPTG), and collecting the broth after 6-h expression. The soluble target protein accounted for 64.6% of the total soluble proteins, and the mature hBD4 expression level was stoichiometrically estimated to be 0.689 g/l. This fusion protein was then purified and cleaved to get the mature hBD4 peptide that showed antimicrobial activity against E. coli and Pseudomonas aeruginosa.
Xu
Zhinan
Z
Institute of Bioengineering, Department of Chemical Engineering and Bioengineering, Zhejiang University (Yuquan campus), Hangzhou, 310027 Zhejiang, People's Republic of China. znxu@zju.edu.cn
Zhong
Zhixia
Z
Huang
Lei
L
Peng
Li
L
Wang
Fang
F
Cen
Peilin
P
D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology
D023181:Antimicrobial Cationic Peptides; C106641:DEFB4A protein, human; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins