High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression

Abstract

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.

Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities

“…The final concentration of IPTG plays an important role in regulating the expression level of recombinant proteins because of its negative effects on cell growth (Xu et al 2006). In this study, four different concentrations of IPTG (0.4, 0.6, 0.8, and 1.0 mM) were examined, and the SDS-PAGE results are shown in Fig.…”

Innate Immunity in the Respiratory Epithelium

“…Although hBD5 and hBD6 have shown antimicrobial activity, they are not expressed in the respiratory epithelium (218)(219)(220). hBD1 is constitutively expressed in the epithelium, whereas hBD2, -3, and -4 can be induced by a variety of bacterial, fungal, and viral pathogens (221)(222)(223)(224)(225)(226)(227).…”

Expression and characterization of codon-optimized Crimean-Congo hemorrhagic fever virus Gn glycoprotein in insect cells

“…To our knowledge, there are no reports about non-optimized Gn expression in insect cells, but some studies have shown that high-level expression of recombinant proteins is affected by factors such as the different codon preferences in different expression systems, and codon optimization can significantly increase the expression level [53][54][55][56]. This is the first report on the cloning and expression of CCHFV Gn glycoprotein in a baculovirus expression system.…”