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KMID : 0545120070170040638
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Journal of Microbiology and Biotechnology
2007 Volume.17 No. 4 p.638 ~ p.343
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Stress-Governed Expression and Purification of Human Type II Hexokinase in Escherichia coli
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Jeong Eun-Ju
Park Kyoung-Sook
Yi So-Yeon
Kang Hyo-Jin
Chung Sang J.
Lee Chang-Soo
Chung Jin-Woong
Seol Dai-Wu
Chung Bong-Hyun
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Abstract
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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 hexahistidinetagged 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 18oC, about 83% of HXK II6×His existed in the soluble fraction, which amounted to a 4.1-fold yield over that expressed at 37oC. The soluble form of HXK II6×His was also highly produced in the presence of 1 M sorbitol under the standard condition (37oC), 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 5 l 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.
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KEYWORD
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Human type II hexokinase, expression, purification, low temperature, osmotic stress
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