title: Efficient production of a soluble fusion protein containing human beta-defensin-2 in E. coli cell-free system.
abstract: Human beta-defensin-2 (hBD2), a small cationic peptide, exhibits a broad range of antimicrobial activity and does not cause microbial resistance. In order to produce hBD2 efficiently, an Escherichia coli cell-free biosynthesis system has been developed as an alternative method. A specific plasmid pIVEX2.4c-trxA-shBD2 was constructed for the cell-free expression of fusion protein (hBD2 linked with His-Tag and Trx-Tag). This allowed enhancement of protein stability and facilitated downstream purification process. Significant amount of target fusion protein was synthesized in the batch-mode bioreactor by optimizing the reaction conditions. About five-fold improvement of productivity (ca. 2.0 mg/ml soluble fusion protein) could be achieved by using a continuous exchange cell-free (CECF) system compared to batch system. One-step affinity chromatographic process was developed to recover high purity fusion protein (95.2%) with overall recovery ratio of about 50%. The fusion protein was cleaved by cyanogens bromide (CNBr), and the mature hBD2 had demonstrated strong inhibition on the growth of E. coli D31 at low concentration.
authors.affiliation: Institute of Bioengineering, Department of Chemical Engineering and Bioengineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Haiqin
authors.initials: H
authors.lastname: Chen
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Xu
authors.forename: Naizheng
authors.initials: N
authors.lastname: Xu
authors.forename: Peilin
authors.initials: P
authors.lastname: Cen
mesh_terms: D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: C106641:DEFB4A protein, human; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins