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논문 기본 정보
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- 한국미생물생명공학회 Journal of Microbiology and Biotechnology Journal of Microbiology and Biotechnology 제17권 제4호
- 발행연도
- 2007.1
- 수록면
- 638 - 643 (6page)
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hexokinase (HXK II) was cloned into the E. coli expressionvector pET 21b and expressed as a C-terminally hexahistidine-taged protein in the BL21 (DE3) strain. The IPTG-inducedHXK I approximately accounted for 17% of the total E. coliproteins, and 81% of HXK II6×His existed in inclusion bodies.To improve the production of soluble recombinant HXK IIprotein, in the functionally active form, we used low temperature,and the osmotic stress expression method. When expressed at18oC, about 83% of HXK II6×His existed in the soluble fraction,which amounted to a 4.1-fold yield over that expressed at37oC. The soluble form of HXK II6×His was also highly producedin the presence of 1 M sorbitol under the standard condition(37oC), which indicated that temperature downshift and lowwater potentials were required to improve the yield of activerecombinant HXK II protein. The expresed protein waspurified by metal chelate affinity chromatography performed2+ ions, resultingin about 40 mg recombinant HXK II protein obtained withpurity over 89% from 5 l of E. coli culture. The identity ofHXK II6×His was confirmed by Western blotting analysis. Takentogether, using the stres-governed expression described inthis study, human active HXK II can be purified in suficientamounts for biochemical and biomedical studies.
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참고문헌 (24)
참고문헌 신청
Arora / 1993 / Structure/function relationships in hexokinase Site-directed mutational analyses and characterization of overexpressed fragments implicate different functions
Birnbaum, M. J / 2004 / On the InterAktion between hexokinase and the mitochondrion 7 : 781 ~ 782
Downward, J / 2003 / le of receptor tyrosine kinases in Gprotein- coupled receptor regulation of Ras: Transactivation or parallel pathways? 376 : e9 ~ 10
I / 1994 / Structural analysis of a protein involved in susceptibility to diabetes : 21925 ~ 21928
Jurgensmeier, J. M / 1998 / Bax directly induces release of cytochrome c from isolated mitochondria 95 : 4997 ~ 5002
Birnbaum, M. J / 2004 / On the InterAktion between hexokinase and the mitochondrion 7 : 781 ~ 782
Downward, J / 2003 / le of receptor tyrosine kinases in Gprotein- coupled receptor regulation of Ras: Transactivation or parallel pathways? 376 : e9 ~ 10
I / 1994 / Structural analysis of a protein involved in susceptibility to diabetes : 21925 ~ 21928
Jurgensmeier, J. M / 1998 / Bax directly induces release of cytochrome c from isolated mitochondria 95 : 4997 ~ 5002
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