title: High-level expression and purification of soluble bioactive recombinant human heparin-binding epidermal growth factor in Escherichia coli.
abstract: Heparin-binding epidermal growth factor (HB-EGF) is a member of highly conserved superfamily of proteins that has potential mitogenic activity and stimulates differentiation and migration of various cell types. Since HB-EGF has three intra-molecular disulfide bonds, a high expression pattern of active HB-EGF in an E. coli expression system was not successfully established. The aim of this study was to increase production of soluble bioactive recombinant human HB-EGF in E. coli by modifying growth conditions and codon optimization. The open reading frame codons of human HB-EGF were optimized to achieve high level expression in E. coli. The optimized codon was amplified, cloned into plasmid pET-32a, and transformed into E. coli BL21 for further expression. The cultivation parameters (temperature and inducer) were optimized to produce a high yield of soluble HB-EGF. The fusion protein was purified by Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Amethylthiazole tetrazolium assay was used to evaluate the bioactivity of the produced recombinant protein. After codon optimization, the codon adaptation index (CAI) was increased from 0.255 in native gene to 0.829 using the optimized sequence. By lowering the temperature to 22°C and the inducer to 0.4 μM, we obtained 35% soluble expression of recombinant and biologically active human HB-EGF. Our data demonstrate that codon optimization increases the yield of HB-EGF in an E. coli expression system. Furthermore, the chosen modifications in cell culturing increase the solubility of recombinant human HB-EGF.
authors.affiliation: Department of Biotechnology, School of Medicine, Arak University of Medical Sciences, Arak, Iran.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Khalili
authors.affiliation: Department of Biotechnology, School of Medicine, Arak University of Medical Sciences, Arak, Iran.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Soleyman
authors.affiliation: Department of Anatomy, School of Medicine, Arak University of Medical Sciences, Arak, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Baazm
authors.affiliation: Institute of Neuroanatomy, Medical Clinic, RWTH Aachen University, Aachen, Germany.
authors.forename: Cordian
authors.initials: C
authors.lastname: Beyer
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D066257:Heparin-binding EGF-like Growth Factor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D003062:Codon; D066257:Heparin-binding EGF-like Growth Factor; D011993:Recombinant Fusion Proteins
keywords: E. coli; codon optimization; heparin-binding EGF; solubility