title: High-level expression of soluble human beta-defensin-2 fused with green fluorescent protein in Escherichia coli cell-free system.
abstract: Human beta-defensin-2 (hBD2), a small cationic peptide, exhibits a broad range of antimicrobial activity and does not acquire any microbial resistance. To produce this uneasily detectable, degradable, and toxic polypeptide efficiently, an alternative approach based on the Escherichia coli cell-free biosynthesis system was proposed. The approach implies that a polypeptide of interest is synthesized as a fusion protein linked to a green fluorescent protein (GFP) through a cleavable spacer. With batch-mode operation, a significant amount of hBD2 fused with GFP (0.25 mg/mL) can be expressed in this cell-free system. The productivity of the fusion protein can be improved up to 1.2 mg/mL by employing a continuous-exchange cell-free system. Furthermore, the GFP moiety provides directly visible and quantitative monitoring of the polypeptide synthesis, and the product is soluble and stable. This work will be helpful in allowing the rapid and visible expression of other similar defensins using an in vitro cell-free system.
authors.affiliation: Institute of Bioengineering, Department of Chemical Engineering and Bioengineering, Zhejiang University, Hangzhou 310027, The People's Republic of China. znxu@zju.edu.cn
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Xu
authors.forename: Haiqin
authors.initials: H
authors.lastname: Chen
authors.forename: Xiufei
authors.initials: X
authors.lastname: Yin
authors.forename: Naizheng
authors.initials: N
authors.lastname: Xu
authors.forename: Peilin
authors.initials: P
authors.lastname: Cen
mesh_terms: D004926:Escherichia coli / Q000502:physiology*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: C106641:DEFB4A protein, human; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins; D049452:Green Fluorescent Proteins