Codon optimization enhances the expression of porcine β-defensin-2 in Escherichia coli

Abstract

ABSTRACT. Porcine β-defensin-2 (pBD2) is a cationic antimicrobial peptide that has therapeutic potential. The amount of pBD2 in nature is limited, and the expression of pBD2 in Escherichia coli is low, probably because a different gene codon is used by prokaryotic organisms to that used by eukaryotes. Codon preference optimization is one of the ways to increase heterologous expression of pBD2. To achieve high expression of pBD2, the pBD2 gene was redesigned according to the preferred codon in E. coli without altering the amino acid sequence. The optimized gene was inserted into expression vector pET-30a and transformed into E. coli BL21 (DE3) plysS. Our results showed that pBD2 was expressed as His-Tag fusion protein at a level that was approximately 4-6 times greater than from the native gene, based on total protein expression. Expressed fusion pBD2 showed antimicrobial activity against both E. coli and Staphylococcus aureus. Moreover, pBD2 showed weak hemolytic activity and strong heat resistance. These results indicate that fusion pBD2 is functional and has similar properties to those of pBD2 from the native gene. Our current study demonstrated that codon optimization could enhance pBD2 expression in E. coli without altering its function. Therefore, the expression of 4979 Codon optimization enhances pBD2 expression in E. coli ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (2): 4978-4988 (2015) pBD2 after codon optimization in heterologous host cells might be useful and is worthy of further research.

Peer Review #1 of "Codon optimization, expression in Escherichia coli, and immunogenicity analysis of deformed wing virus (DWV) structural protein (v0.1)"

“…, and VP3 sequences of the DWV LN8/17 strain were analyzed and optimized based on E. coli-preferred codons without changing the amino acid sequence of the corresponding proteins (Gao et al, 2015;Mansouri et al, 2013;Wang et al, 2012). High-frequency-usage codons in E. coli were the most commonly used for each of the individual amino acids (Tian et al, 2017).…”

Evaluation of three different vectors on human iLRP expression in Escherichia coli

“…Codon optimization can refer to several meanings as, avoiding rare codons with low utilization, simplifying mRNA's secondary structure after gene transcription, removing the motif which is not conducive to efficient expression and add the helpful one, adjusting the GC content and other methods to re-design genes [8,9]. In this study, GC content, codon adaptation index (CAI), mRNA structure and cis-acting elements in the human iLRP gene were optimized to match requirements for the expression in Escherichia coli (E. coli) by NG®Codon Optimization Technology.…”

Preparation of polyclonal antibody against porcine beta defensin 2 and identification of its distribution in tissues of pig

“…Based on BL21(DE3)-pET-pBD2, which was constructed in our lab, his-pBD2 was expressed and purified as previously described (Li et al, 2013a;Gao et al, 2015). The molecular weight of purified his-pBD2 was about 12 kDa, and the purity was greater than 90% according to Gel-ProAnalyzer (4.0) analysis (Figure 1).…”

“…His-pBD2 was expressed by inducing the Escherichia coli strain BL21(DE3)-pET-pBD2, which was constructed in our laboratory, with isopropyl β-D-1-thiogalactopyranoside (IPTG), and the protein was purified as previously described (Li et al, 2013a;Gao et al, 2015). The purified his-pBD2 was analyzed by SDS-PAGE, and used for the preparation of antibodies.…”

“…Thus, in this study, we prepared and characterized an anti-pBD2 polyclonal antibody. To generate an anti-pBD2 polyclonal antibody, we used a previously described strain, BL21(DE3)-pET-pBD2 (Gao et al, 2015), to express a histagged pBD2 (his-pBD2) fusion protein. Polyclonal antibody was prepared by injecting purified hispBD2 into New Zealand white rabbits.…”