title: Examination of alternate codon bias solutions for expression and purification of recombinant mechano-growth factor in Escherichia coli.
abstract: Mechano-growth factor (MGF), an alternative splicing variant of insulin-like growth factor-1 (IGF-1) gene, promotes cell proliferation and inhibits cell differentiation. It also plays an important role in tumor development. It is important to optimize the production process and achieve MGF protein because there is no commercial MGF protein available. In this study, the human MGF gene is cloned into pGEX-4T-1 and the recombinant human MGF (rhMGF) protein could be expressed in Rosetta (DE3) by isopropyl β-D-1-thiogalactopyranoside induction but not in BL21 (DE3). Mutation from rare codons to Escherichia coli preferred ones is performed. We obtain MGF(Mut54-56) and MGF(Mut-total) fragments through site-directed mutagenesis and overlapping PCR. Both pGEX-4T-1/MGF(Mut54-56)- and pGEX-4T-1/MGF(Mut-total)-transformed BL21 (DE3) can be induced to express rhMGF protein. To optimize the production technology, expression and purification of rhMGF are analyzed and compared in Rosetta (DE3) and BL21 (DE3). Results indicate that rhMGF expression in BL21 (DE3) is significantly higher than that in Rosetta (DE3). The protein yield of pGEX-4T-1/MGF(Mut-total) in BL21 (DE3) is higher than that of pGEX-4T-1/MGF(Mut54-56). We test the biological activity of MGF protein purified by affinity chromatography in C2C12 cell line and find that rhMGF promotes cell proliferation significantly. In conclusion, we establish a method to produce rhMGF economically with high biological activity in BL21 (DE3).
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, People's Republic of China.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Feng
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, People's Republic of China.
authors.forename: Rongxue
authors.initials: R
authors.lastname: Wan
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, People's Republic of China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Yi
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, People's Republic of China.
authors.forename: Ling
authors.initials: L
authors.lastname: He
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.affiliation: Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, People's Republic of China.
authors.forename: Liling
authors.initials: L
authors.lastname: Tang
mesh_terms: D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I
keywords: mechano-growth factor; protein expression and purification; recombinant protein; site-directed mutagenesis