Santi D V, Edman U, Minkin S, Greene P J
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Protein Expr Purif. 1991 Oct-Dec;2(5-6):350-4. doi: 10.1016/1046-5928(91)90093-x.
Catalytically active Pneumocystis carinii thymidylate synthase is expressed to the extent of about 4% of the soluble protein in Escherichia coli chi 2913 harboring plasmid pUETS-1.8 (U. Edman, J. C. Edman, B. Lundgren, and D. V. Santi, Proc. Natl. Acad. Sci. USA 86, 6503-6507, 1989). Ion-exchange, affinity, hydrophobic, and reactive dye agarose chromatography steps were explored to devise a large-scale purification protocol for P. carinii thymidylate synthase. Sequential DE52, Q-Sepharose, phenyl-Sepharose, and Cibacron Blue F3GA chromatography yielded enzyme that was homogeneous by SDS-PAGE in a yield of over 50%. The sequence of the first 10 amino acid residues of the purified protein was in accord with that predicted from the DNA sequence. Isoelectric focusing gave a pI of 6.2. Kinetic analysis of the purified enzyme revealed that the Km values were 4.7 +/- 1.3 microM for dUMP and 15.7 +/- 4.3 microM for 5,10-methylenetetrahydrofolate, similar to those of many other thymidylate synthases; the kcat of the most active preparation was 0.8 s-1. The enzyme is stable for at least 2 months when stored at -80 degrees C in the presence of 40% glycerol, Tris-HCl, and thiol.
在携带质粒pUETS - 1.8的大肠杆菌chi 2913中，具有催化活性的卡氏肺孢子虫胸苷酸合成酶的表达量约为可溶性蛋白的4%（U. 埃德曼、J. C. 埃德曼、B. 伦德格伦和D. V. 桑蒂，《美国国家科学院院刊》86, 6503 - 6507, 1989）。人们探索了离子交换、亲和、疏水和活性染料琼脂糖层析步骤，以设计出一种大规模纯化卡氏肺孢子虫胸苷酸合成酶的方案。依次通过DE52、Q - 琼脂糖凝胶、苯基 - 琼脂糖凝胶和Cibacron Blue F3GA层析得到的酶，经SDS - PAGE分析显示为均一性，产率超过50%。纯化蛋白的前10个氨基酸残基序列与从DNA序列预测的序列一致。等电聚焦给出的pI为6.2。对纯化酶的动力学分析表明，其对dUMP的Km值为4.7±1.3微摩尔，对5,10 - 亚甲基四氢叶酸的Km值为15.7±4.3微摩尔，这与许多其他胸苷酸合成酶的Km值相似；活性最高的制剂的kcat为0.8 s-1。当在40%甘油、Tris - HCl和硫醇存在的情况下于-80℃储存时，该酶至少稳定2个月。
