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Calorimetric study on thermally-induced conformational change of dihydrofolate reductase

Chinese Science Bulletin 1996年第21期41卷 1802-1805页

作者：刘云娜谈夫 Institute of Chemistry Chinese Academy of Sciences Beijing 100080 China

dihydrofolate reductase(DHFR)(EC 1.***.1.3)exists in all *** is an importantcomponent of the chemistry of DNA synthesis in ***,a potent inhibitor ofDHFR,is a well-known anticancer *** has been employed extensively in ***,the study on the conformational change of DHFR gives occasion topeople’s close *** thermally-induced conformational change of DHFR

关键词： dihydrofolate reductase conformational change calorimetry.

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Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

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Science China(Life Sciences) 1997年第2期40卷 202-209页

作者：范映辛朱笠周筠梅邹承鲁 State Key Laboratory of Biomacromolecules Institute of Biophysics Chinese Academy of Sciences Beijing 100101 China

The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hy-drochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteol-ysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conforma-tional change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0.75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at the active site first, until to a critical concentration of the denaturant the overall conformation change of the molecule may occur cooperatively. Activation of DHFR in low GuHCl concentration is due to the subtle conformational change at the active site of the enzyme. Although the conformational change at the active site decreases the affinity of the enzyme for the substrates, it accelerates the velocity of the limiting step in the catalytic reaction, i.e. the velocity of the disso-ciation of products from the ternary complex.

关键词： dihydrofolate reductase guanidine hydrochloride activation protein unfolding.

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High expression of EPO gene using un-dhfr negative cell

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Chinese Science Bulletin 1998年第23期43卷 1996-2001页

作者： Zhenzhong Cui Pengpeng Liu Shunzhang Qi Heng Shen China Agr Univ Coll Biol Sci Dept Biochem Beijing 100094 Peoples R China

Erythropoietin （EPO） genomic gene was cloned and its expression vector pOP13/EPO was constructed. CHOK12 cell was transfected by this vector using lipofectin method. A stable expression cell strain C10 cell with the EPO production at 160IU/d in 10\+6 cells were obtained at 400 μg/mL G418. Based on the C10 cell, another vector pHY/dhfr （dihydrofolate reductase） that carries a dhfr gene and a selecting marker of hygromycin B resistant gene was transferred to this cell. Several cell clones were obtained at 200 μg/mL hygromycin B. These cell clones that can express both EPO gene and exogenous dhfr gene were selected under the progressively increased concentration to 1 μmol methotrexate（MTX）. Some high EPO expression cell clones were obtained, the highest expression was 2 400 IU/d in 10\+6 cells, 15 times higher than that without MTX pressure. Then, a method of EPO high expression by using undhfr negative cell was primarily established. EPO bioactivity was found by using TF1 cell.

关键词： erythropoietin dihydrofolate reductase gene methotrexate un_dhfr negative cell cell expression.

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