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| Chicken (Gallus gallus) HNF1α Expression in Escherichia coli and Its Purification |
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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.
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Received: 10 July 2017
Published: 14 February 2018
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