Production of Bioactive Human β-defensin-3 in Escherichia coli by Soluble Fusion Expression

Abstract

A codon optimized mature human beta-defensin-3 gene (smHBD3) was synthesized and fused with TrxA to construct pET32-smHBD3 vector, which was transformed into E. coli BL21(DE3) and cultured in MBL medium. The volumetric productivity of fusion protein reached 0.99 g fusion protein l(-1), i.e. 0.21 g mature HBD3 l(-1). Ninety-six percentage of the fusion protein was in a soluble form and constituted about 45% of the total soluble protein. After cell disruption, the soluble fusion protein was separated by affinity chromatography and cleaved by enterokinase, and then the mature HBD3 was purified by cationic ion exchange chromatography. The overall recovery ratio of HBD3 was 43%. The purified mature HBD3 demonstrated antimicrobial activity against E. coli.

Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins

“…In contrast to earlier reports about defensin production in E. coli K-12 strains (Huang et al, 2006;Pazgier and Lubkowski, 2006;Xu et al, 2006), this study reports for the first time the production of active HBD2 defensin in a probiotic E. coli strain (Nissle 1917), which is licensed as a drug for the treatment of patients suffering from e.g. diarrhea or ulcerative colitis.…”

“…It has been reported that a fusion expression system with thioredoxin (TrxA) resulted in an increased yield of soluble products (LaVallie et al 2000). This has been observed for HBD2 and HBD3 (Huang et al, 2006;Xu et al, 2006). …”

Production and purification of recombinant hypocholesterolemic peptides

“…Escherichia coli has been used to produce small peptides composed of 21-50 amino acid residues (Huang et al 2007;Rao et al 2005). However, there has been no report on the large-scale production of a small peptide with fewer than 10 amino acid residues.…”

Enhanced Expression and Primary Purification of Soluble HBD3 Fusion Protein in Escherichia coli

“…Thus, soluble expression strategies are preferred to produce functional proteins efficiently, avoiding the refolding procedure. In our previous work, soluble fusion protein containing human β-defensin-2 (HBD2) and HBD3 were successfully expressed after codon optimization in E. coli as well as the cell-free system, and the purified mature HBD2 and HBD3 were fully bioactive [11][12][13][14][15][16][17]. However, the productivity (0.99 g/L) of HBD3 fusion protein in our constructed recombinant E. coli strain [12] was relatively low.…”

“…In our previous work, soluble fusion protein containing human β-defensin-2 (HBD2) and HBD3 were successfully expressed after codon optimization in E. coli as well as the cell-free system, and the purified mature HBD2 and HBD3 were fully bioactive [11][12][13][14][15][16][17]. However, the productivity (0.99 g/L) of HBD3 fusion protein in our constructed recombinant E. coli strain [12] was relatively low. Due to the a relatively low recovery rate of mature human beta-defensin 3 after a multi-step down-stream purification process, a high-level expression of this HBD3 fusion protein is preferred for the large-scale preparation of mature recombinant HBD3.…”