Stress-governed expression and purification of human type II hexokinase in Escherichia coli.
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 II(6xHis) 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 degrees C, about 83% of HXK II(6xHis) existed in the soluble fraction, which amounted to a 4.1-fold yield over that expressed at 37 degrees C. The soluble form of HXK II(6xHis) was also highly produced in the presence of 1 M sorbitol under the standard condition (37 degrees 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 5 l of E. coli culture. The identity of HXK II(6xHis) 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.
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-600, Korea.
Eun-Ju
EJ
Jeong
Kyoungsook
K
Park
So Yeon
SY
Yi
Hyo-Jin
HJ
Kang
Sang J
SJ
Chung
Chang-Soo
CS
Lee
Jin Woong
JW
Chung
Dai-Wu
DW
Seol
Bong Hyun
BH
Chung
Moonil
M
Kim
D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006593:Hexokinase / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009997:Osmotic Pressure; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012995:Solubility; D013696:Temperature
D011994:Recombinant Proteins; D006593:Hexokinase