Objective To optimize the conditions for the induction expression and purification of recombinant Hq012 (rHq012) inclusion bodies of Haemaphysalis qinghaiensis.Methods The novel gene Hq012 with no homogenous sequences in the GenBank database was cloned from the cDNA expression library of H. qinghaiensis to be transfected into Escherichia coli Rosetta (DE3) using the prokaryotic expression plasmid pET-30a-Hq012. Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of rHq012 protein. After homogenizing the bacteria under high pressure, the inclusion bodies were dissolved in 6 mol/L guanidine hydrochloride solution. We compared the yield of the target protein obtained by first renaturation and then purification (refolding by dilution followed by purification with nickel ion affinity chromatography) and by first purification and then renaturation (purification with nickel ion affinity chromatography followed by refolding by dialysis). The obtained protein was identified by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis.Results The nucleotide sequence length of the Hq012 gene was 759 bp, containing an open reading frame of 486 bp, encoding a protein with a relative molecular mass of 18 500. The protein expression was the highest at 8-hour induction with IPTG at 0.1 mmol/L. The inclusion bodies were dissolved with 6 mol/L guanidine hydrochloride. The yield of rHq012 harvested by first renaturation and then purification was higher compared with that by first purification and then renaturation. The relative molecular mass of the protein purified by nickel ion affinity chromatography was about 17 000, which was consistent with expected results.Conclusions Induction at an concentration of 0.1 mol/L IPTG for 8 hours was the best induction condition, and the method of first refolding followed by purification was an optimal purification method for the tick-derived recombinant protein rHq012. The study provides a reference for the purification of inclusion body protein.
MA Jing, CHEN Kai-ting, GUANG Hui, ZHAO Wen-bin, YANG Yin-ran, GAO Jin-liang, CAO Mei-na
. Expression and purification of Hq012 gene of Haemaphysalis qinghaiensis in Escherichia coli[J]. Chinese Journal of Vector Biology and Control, 2023
, 34(6)
: 713
-718
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DOI: 10.11853/j.issn.1003.8280.2023.06.001
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