Production and characterization of bioactive recombinant resistin in Escherichia coli.
Type 2 diabetes, characterized by peripheral target tissue resistance to insulin, is epidemic in industrialized countries and is strongly associated with obesity. The protein hormone, resistin, secreted specifically by the adipose tissues, is found to antagonize insulin action upon glucose uptake and may serve as an important role between human obesity and insulin resistance. Here, we report the production of bioactive recombinant resistin in Escherichia coli. cDNA of resistin was obtained by RT-PCR from mRNA of mouse differentiated NIH/3T3-L1 cells. The cDNA of mature resistin was inserted in the pQE-31 vector and the recombinant plasmid was transferred into E. coli JM109. After IPTG induction, the rec. resistin found in the inclusion body was dissolved in 6 M guanidine-HCl in the presence of 10 mM beta-mercaptoethanol. The His-tag containing protein was purified by Ni-NTA column to 95% homogeneity. After a quasi-static-like refolding process, the secondary structure of the rec. resistin was elucidated by circular dichroism which indicated that the protein was composed of 34.3% alpha-helix, 8.9% beta-sheet, 23.4% beta-turn, and 31.2% unordered structure. No disulfide-linked homodimers were formed in SDS-PAGE analysis under non-reducing conditions. The rec. resistin showed a dose-dependent antagonizing action against insulin in [3H]-2-deoxy-glucose transport in a broad range from 1 ng ml(-1) to 10 microg ml(-1) of resistin. A suppression of 85% of transport was achieved at the dosage of 10 microg ml(-1). This result may indicate that the rec. resistin does not need to form homodimers to establish its bioactivity. The rec. resistin will be useful for exploring the biological functions of this newly discovered hormone.
Juan
Chi-Chang
CC
Department of Medical Research and Education, Veterans General Hospital, Taiwan, Taipei, China
Kan
Lou-Sing
LS
Huang
Cheng-Chih
CC
Chen
Shih-Shih
SS
Ho
Low-Tone
LT
Au
Lo-Chun
LC
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002942:Circular Dichroism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D005947:Glucose / Q000493:pharmacokinetics; D006729:Hormones, Ectopic / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D041681:NIH 3T3 Cells; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D052243:Resistin
D006729:Hormones, Ectopic; D007328:Insulin; D011994:Recombinant Proteins; D052243:Resistin; C499623:Retn protein, mouse; D005947:Glucose