Trujillo M, Donald R G, Roos D S, Greene P J, Santi D V
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
Biochemistry. 1996 May 21;35(20):6366-74. doi: 10.1021/bi952923q.
We have expressed catalytically active Toxoplasma gondii dihydrofolate-thymidylate synthase (DHFR-TS) and the individual TS and DHFR domains in Escherichia coli using the T7 promoter of pET-15b. DHFR-TS constituted approximately 10% of the total soluble cell protein and was purified using methotrexate-Sepharose chromatography to yield 10 mg of homogeneous DHFR-TS per liter of culture. The DHFR domain was recovered as insoluble inclusion bodies which could be unfolded and refolded to recover soluble, active enzyme. The TS domain was overexpressed as a soluble protein by growing the cells at 24 degrees C; this is the first report of the expression of an active TS domain from a bifunctional enzyme. The kcat and K(m) values for DHFR-TS are similar to those of other previously characterized protozoan DHFRs and TSs. The antimicrobial antifolates, TMP and Pyr, inhibit DHFR activity of the bifunctional protein in accord with their effects in crude enzyme preparations and in vivo systems. Kinetic parameters and Ki values for TMP and Pyr with the isolated DHFR domain were identical to the values for DHFR in the bifunctional enzyme. Evidence of kinetic channeling of the dihydrofolate product of TS to the DHFR domain in the bifunctional enzyme was obtained by kinetic and inhibition studies. Properties such as yield, stability, and activities of the recombinant T. gondii DHFR-TS provide clear advantages over other bifunctional DHFR-TSs as a model for future studies.
我们利用pET - 15b的T7启动子在大肠杆菌中表达了具有催化活性的弓形虫二氢叶酸 - 胸苷酸合酶（DHFR - TS）以及单独的TS和DHFR结构域。DHFR - TS约占总可溶性细胞蛋白的10%，通过甲氨蝶呤 - 琼脂糖凝胶层析进行纯化，每升培养物可获得10 mg的纯DHFR - TS。DHFR结构域以不溶性包涵体形式回收，可通过变性和复性来恢复可溶性的活性酶。通过在24℃培养细胞，TS结构域作为可溶性蛋白得以过量表达；这是关于从双功能酶中表达活性TS结构域的首次报道。DHFR - TS的kcat和Km值与其他先前已表征的原生动物DHFR和TS的值相似。抗菌抗叶酸剂甲氧苄啶（TMP）和乙胺嘧啶（Pyr）抑制该双功能蛋白的DHFR活性，这与它们在粗酶制剂和体内系统中的作用一致。TMP和Pyr与分离的DHFR结构域的动力学参数和Ki值与双功能酶中DHFR的值相同。通过动力学和抑制研究获得了双功能酶中TS的二氢叶酸产物向DHFR结构域进行动力学通道化的证据。重组弓形虫DHFR - TS的产量﹑稳定性和活性等特性，相较于其他双功能DHFR - TS，为未来研究提供了明显优势。
