Abstract:Hepatocyte nuclear factor 1α (HNF1α) plays an important role in the carbohydrate and lipid metabolism by regulating numerous related genes in liver on mammals, but there are few studies in poultry. To obtain the chicken (Gallus gallus) HNF1α protein, the CDS fragment of chicken HNF1α was amplified by PCR from the plasmid pcDNA3.1-HNF1α constructed previously in our lab. After the fragment being cloned into the site between NdeⅠ and XhoⅠ of the pET30a, which protein fused 6-His at its C-terminus, the BL21-pET30a-HNF1α recombinant expression strain was successfully constructed through the recombinant expression plasmid (pET30a-HNF1α) being transformed into BL21 (DE3). To induce the HNF1α recombinant protein, the BL21-pET30a-HNF1α was cultured in Luria-Bertani (LB) medium with different concentrations of isopropyl-β-D-thiogalactoside (IPTG) (such as 0.1, 0.2, 0.4, 0.8 mmol/L). The analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and Western blot showed that the recombinant protein was more highly expressed in BL21 at 37 ℃ for 4 h with 0.1 mmol/L IPTG than others. And it's molecular weight was about 69 kD, which was consistent with molecular weight by theoretical calculation. The results showed that the recombinant protein was mainly expressed with the form of inclusion body although the inducible temperature was dropped to 15 ℃. Then after these inclusion bodies were completely denatured with 8 mol/L urea, the HNF1α recombinant proteins were adsorbed onto the Ni2+-IDA resin by 6-His tag fused at its C- terminal, and these proteins were refolded in situ by the urea being phase out to zero in the washing process. Finally, the refolded proteins were eluted by the elution buffer with 300 mmol/L iminazole. By the SDS-PAGE gel analysis, the results showed that the chicken HNF1α recombinant protein near 95% purity was obtained from the denaturing solution using this one-step method combined with Ni2+ affinity chromatography and solid-fluid renaturation in situ. These results lay a foundation for the further study of the structure and function of the HNF1α, simultaneously pave the way for the preparation of the corresponding antibody.
[1]Cereghini, S.Liver-enriched transcription factors and hepatocyte differentiation[J].FASEB journal, 1996, 10(2):267-282[2]Pontoglio M., BarraJ., Hadchouel M., et al.Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome[J].Cell, 1996, 84(4):575-585[3]Yoshiuchi I, Yamagata K, Yang Q, et al.Three new mutations in the hepatocyte nuclear factor-1alpha gene in Japanese subjects with diabetes mellitus: clinical features and functional characterization[J].Diabetologia, 1999, 42(5):621-626[4]Mitchell SM, Frayling TM. The role of transcriptionfactors in maturity-onset diabetes of the young[J].Molecular Genetic Metabolism, 2002, 77(1-2):35-43[5]Shih DQ, Bussen M, Sehayek E, et al. Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism[J].Nature Genetics, 2001, 27(4):375-382[6]Pontoglio M, Sreenan S, Roe M, et al.Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice[J].The journal of Clinical Investigation,, 1998, 101(10):2215-2222[7]St-Jean M, Boudreau F, Carpentier AC, et al.HNF1α defect influences post-prandial lipid regulation[J]., 2017, 12(5):-[8]Pedersen KB, Chhabra KH, Nguyen VK, et al. The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs[J].Biochimica et biophysica acta, 2013, 1829(11):1225-1235[9]Niu M-J, Yang J-K, Lin S-S, et al. Loss of angiotensin-converting enzyme 2 leads to impaired glucose homeostasis in mice[J].Endocrine, 2008, 34(1-3):56-61[10]Yamagata K.Regulation of pancreatic beta-cell function by the HNF transcription network: lessons from maturity-onset diabetes of the young (MODY)[J].Endocrine journal, 2003, 50(5):491-499[11]Armendariz AD, Krauss RM.Hepatic nuclear factor 1-alpha: inflammation, genetics, and atherosclerosis[J].Current opinion lipidology, 2009, 20(2):106-111[12]Li H, Dong B, Park SW, et al.Hepatocyte nuclear factor 1alpha plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine[J].The journal of biology Chemistry, 2009, 284(42):28885-28895[13]Shende VR, Wu M, Singh AB, et al.Reduction of circulating PCSK9 and LDL-C levels by liver-specific knockdown of HNF1α in normolipidemic mice[J].Journal of lipid research, 2015, 56(4):801-809[14]Sucajtys-Szulc E, Szolkiewicz M, Swierczynski J, et al.Up-regulation of Hnf1α gene expression in the liver of rats with experimentally induced chronic renal failure - A possible link between circulating PCSK9 and triacylglycerol concentrations[J].Atherosclerosis, 2016, 248:17-26[15]Hussain MM, Shi J, Dreizen P.Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly.[J].Journal of lipid research, 2003, 44(1):22-32[16]Dai K, Hussain MM. NR2F1 disrupts synergistic activation of the MTTP gene transcription by HNF-4α and HNF-1α[J].Journal of lipid research, 2012, 53(5):901-908[17]Armendariz AD, Krauss RM.Hepatic nuclear factor 1-alpha: inflammation, genetics, and atherosclerosis.[J].Current Opinion lipidology, 2009, 20(22):106-111[18]Chi YI, Frantz JD, Oh BC, et al.Diabetes mutations delineate an atypical POU domain in HNF-1alpha[J].Molecular cell, 2002, 10(5):1129-1137[19]于淼, 杨晓明.HNF1α的蛋白质复合体[J].医学分子生物学杂志, 2007, 4(4):335-338[20]Grajer KH, H?rlein A, Igo-Kemenes T.Hepatic nuclear factor 1 alpha (HNF-1 alpha) is expressed in the oviduct of hens and interacts with regulatory elements of the lysozyme gene.[J].Biological chemistry Hoppe-Seyler, 1993, 375(5):319-326[21]解庭波.大肠杆菌表达系统的研究进展[J].长江大学学报(自然科学版), 2008, 5(3):77-82[22]Crivelli E, Cardamone M, Puri NK.A single step method for the solubilization and refolding of recombinant protein from E. coli inclusion bodies[J].Australian journal biotechnology, 1991, 5 (2):78-80,86