title: PEGylation-aided refolding of globular adiponectin.
abstract: Globular adiponectin (GAD) as the active domain of adiponectin is a promising candidate for anti-diabetic drug development. The recombinant production of GAD in Escherichia coli, however, is difficult because it is mainly expressed as inclusion bodies which need to be refolded to regain function. In this study we developed a novel method for refolding of GAD with a high efficiency by using polyethylene glycol (PEG) conjugation. An artificially designed DNA sequence encoding for GAD was synthesized and inserted into the pET28a vector to construct an expression plasmid which was thereafter transformed into E. coli BL21 (DE3) host cells for heterologous expression. After bacterial cell culture employing auto-induction medium, the inclusion bodies were collected, washed and dissolved in guanidine hydrochloride before PEG conjugation. Then the PEG-conjugated GAD was refolded by dialysis and purified by two steps of chromatography. The refolded conjugate showed a marked glucose-lowering activity in mice, demonstrating that it had been successfully refolded. As a convenient method, PEGylation-aided refolding could also be tested on other proteins to explore its suitability.
authors.affiliation: State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Gao
authors.forename: Yue
authors.initials: Y
authors.lastname: Tong
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Gao
authors.forename: Wenbing
authors.initials: W
authors.lastname: Yao
mesh_terms: D052242:Adiponectin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005951:Glucose Tolerance Test; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry*; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
chemical_list: D052242:Adiponectin; D007004:Hypoglycemic Agents; D011993:Recombinant Fusion Proteins; D011092:Polyethylene Glycols