title: Enhanced expression and primary purification of soluble HBD3 fusion protein in Escherichia coli.
abstract: Human beta-defensin-3(HBD3) is a low molecular weight cationic peptide with a broad antimicrobial spectrum. A recombinant Escherichia coli (pET32-smHBD3) was constructed to produce HBD3 fusion protein (TrxA-HBD3) before, but the productivity is relatively low. In the present work, the effects of different expression conditions were systematically investigated to improve the expression level of the fusion protein. With regard to the volumetric productivity, the optimal conditions were determined as follows: cultivation at 34 degrees C in MBL medium, induction at middle stage of the exponential growth phase with 0.4 mM isopropylthio-D-galactoside, and postinduction expression for 8 h. Under these conditions, the volumetric productivity of the fusion protein reached 2.55 g/L, i.e., 0.55 g mature HBD3/L, which was about 2.6 times of that obtained under the unoptimized conditions. And the target protein still maintained high solubility (> or =97.9%) and accounted for 66% of the total soluble protein. A cationic exchange purification step was employed to obtain high-purity target protein (90%) with a recovery ratio of 78%. This soluble expression level of HBD3 fusion protein was the highest among all the reported literature and facilitated the development of high efficient purification of HBD3.
authors.affiliation: Institute of Bioengineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Xu
authors.forename: Zhixia
authors.initials: Z
authors.lastname: Zhong
authors.forename: Li
authors.initials: L
authors.lastname: Peng
authors.forename: Haiqin
authors.initials: H
authors.lastname: Chen
authors.forename: Peilin
authors.initials: P
authors.lastname: Cen
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility; D013696:Temperature; D023083:beta-Defensins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C422950:DEFB103A protein, human; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins; D007544:Isopropyl Thiogalactoside