Abstract：As a recently discovered muscle factor, human(Homo sapiens) Irisin is an excretive polypeptide fragment whose precursor is fibronectin type Ⅲ domain-containing protein5(FNDC5). Irisin can transfer the signal of skeletal muscle and keep the relationship between skeletal muscle and the peripheral tissues. Irisin will certainly be a very promising active factor, and become the new targets for prevention and control of metabolic disease and its complications. Therefore, exploiting a method that can be used to mass produce and easily purify human Irisin recombinant protein is very significant. To achieve the prokaryotic expression of human Irisin, the FNDC5 cDNA was obtained from the muscles, then the restriction enzyme cutting site NdeⅠ and XhoⅠ were introduced to human Irisin gene by the specific primers amplification. The gene sequence was digested by NdeⅠ and XhoⅠ and cloned into prokaryotic expression vector, pET-30a that digested by NdeⅠ and XhoⅠ, to form pET-30a-Irisin. The recombinant plasmid was transformed to Escherichia coli Rosetta (DE3) pLysS and was screened by kanamycin to form Rosetta-pET-30a-Irisin recombination strain. Then the expression of interest protein was induced with 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol/L isopropyl β-D-1- thiogalactopyranoside(IPTG), and the recombinant protein detected by sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE), the gommures was stained by Coomassie brilliant blue for 4 hours and discolored for 3 hours to analyze the expression of recombinant proteins. In order to realize the soluble expression of recombinant proteins, the Rosetta-pET-30a-Irisin recombination strain was induced with 0.1 mmol/L IPTG at 18 ℃. And the recombination strains induced with IPTG were broken by ultrasonication, the soluble recombinant proteins in cracked supernatant were purified by Ni-sepharose, then detected and identified by Western blot. The DNA sequence analysis showed, that the sequence of cloned human Irisin was in accord with that published by NCBI, and Escherichia coli recombinant expression vector pET-30a-Irisin had been successfully constructed. The results of SDS-PAGE and Western blot indicated that, the Escherichia coli recombination strain Rosetta-pET-30a-Irisin could realize the dissoluble expression of human Irisin induced with 0.1 mmol/L IPTG at 18 ℃. The 14 kD recombinant protein was purified by Ni-sepharose, and the recovery rate was about 2 mg/L. A soluble expression system for human Irisin protein and high purity of human Irisin protein was successfully constructed and obtained. And the system was simple operation and low cost. This study provides theoretical references for the study of structure, function and application of human Irisin.
[1]Bostr?m P1, Wu J, Spiegelman BM, et al.A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis[J].Nature, 2012, 481(7382):463-468[2]Teufel A, Malik N, Westpha H et al.Frcp1 and Frcp2,two novel fibronectin type III repeat containing genes[J].Gene, 2002, 297(1-2):79-83[3] Andreu Ferrer-Martínez, Pilar Ruiz-Lozano, Kenneth R, Chien et al. .Mouse PeP: A novel peroxisomal protein linked to myoblast differentiation and development[J].[J].Dev Dyn. ., 2002, 224(2):154-167[4] Dun SL, Lyu RM, Chen YH, et a1．.Irisin-immunoreactivity in neural and non-neural cells of the rodent[J][J].Neuroscience,, 2013, 240:155-162[5]Ferrer-Martínez A, Ruiz-Lozano P, Chien KR.Mouse PeP: a novel peroxisomal protein linked to myoblast differentiation and development[J].Dev Dyn, 2002, 224(2):154-167[6]Spiegelman BM.Banting Lecture 2012: Regulation of adipogenesis: toward new therapeutics for metabolic disease[J].Diabetes, 2013, 62(6):1774-1782[7]Kelly DP.Medicine,Irisin,light my fire[J].Science, 2012, 336(6077):42-43[8]Komatsu M, Tong Y, Aoyama T, et al.Multiple roles of PPARalpha in brown adipose tissue under constitutive and cold conditions[J].Genes Cells, 2010, 15(2):91-100[9]Villarroya F.Irisin,turning up the heat[J].Cell Metab, 2012, 15(3):277-278[10]Castillo-Quan JI.From white to brown fat through the PGC-1α-dependent myokine irisin: implications for diabetes and obesity[J].Dis Model Mech, 2012, 5(3):293-295[11]Joo Young Huh, Grigorios Panagiotou, Vassilis Mougios, et al.FNDC5 and irisin in humans: IPredictors of circulating concentrations in serum and plasma and II. mRNA expressionand circulating concentrations in response to weight loss andexercise[J].Metabolism, 2012, 61(12):1725-1738[12] AI-Daghri NM, Alkharfy KM, Rahman S, et a1.Irisin as a predictor of glucose metabolism in children: sexually dimorphic effects[J]. [J].Eur J Clin Invest, 2014,, 44(2):119-124[13]Sesti G, Andreozzi F, Perticone F, et al.High circulating irisin levels are associated with insulin resistance and vascular atherosclerosis in a cohort of nondiabetic adult subjects[J].Acta Diabetol, 2014, 51(5):705-713[14] Stengel A, Hofmann T, Goebel- Stengel M, et al.Circulating levels of irisin in patients with anorexia nervosa and different stages of obesity-correlation with body mass index[J].[J].Peptides, 2013, 39:125-130[15] Zhang HJ, Zhang XF, Ma ZM, et al,. Irisin is inversely associated with intrahepatic triglyceride contents in obese adults[J].Journal of Hepatol, 2013, 59(3):557-562[16]Lecker SH, Zavin A, Cao P, et al.Expression of the irisin precursor FNDC5 in skeletal muscle correlates with aerobic exercise performance in patients with heart failure[J].Circulation: Heart Failure, 2012, 5(6):812-818[17]Gustafsson C, Govindarajan S, Minshull J.Codon bias and heterologous protein expression[J].Trends Biotechnol, 2004, 22(7):346-53
