Stress-governed expression and purification of human type II hexokinase in Escherichia coli

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Stress-governed expression and purification of human type II hexokinase in Escherichia coli
Eun-Ju Jeong; Kyoungsook Park; So Yeon Yi; Hyo-Jin Kang; Sang Jeon Chung; Chang-Soo Lee; Jin Woong Chung; D W Seol; Bong Hyun Chung; Moonil Kim
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 17, no. 4, pp. 638-643
Publication Year
The full encoding sequence for human type II hexokinase (HXK II) was cloned into the E. coli expression vector pET 21b and expressed as a C-terminally hexahistidine-tagged protein in the BL21 (DE3) strain. The IPTG-induced HXK II approximately accounted for 17% of the total E. coli proteins, and 81% of HXK II6×His existed in inclusion bodies. To improve the production of soluble recombinant HXK II protein, in the functionally active form, we used low temperature, and the osmotic stress expression method. When expressed at 18°C, about 83% of HXK II6×His existed in the soluble fraction, which amounted to a 4.1-fold yield over that expressed at 37°C. The soluble form of HXK II6×His was also highly produced in the presence of 1 M sorbitol under the standard condition (37°C), which indicated that temperature downshift and low water potentials were required to improve the yield of active recombinant HXK II protein. The expressed protein was purified by metal chelate affinity chromatography performed in an IDA Excellose column charged with Ni2+ ions, resulting in about 40 mg recombinant HXK II protein obtained with purity over 89% from 51 of E. coli culture. The identity of HXK II6×His was confirmed by Western blotting analysis. Taken together, using the stress-governed expression described in this study, human active HXK II can be purified in sufficient amounts for biochemical and biomedical studies.
expressionhuman type II hexokinaselow temperatureosmotic stresspurification
Korea Soc-Assoc-Inst
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Division of Research on National Challenges > Bionanotechnology Research Center > 1. Journal Articles
Critical Diseases Diagnostics Convergence Research Center > 1. Journal Articles
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