Cloning, expression, and characterization of Cryptococcus neoformans dihydrofolate reductase.
Cloning, expression, and characterization of Cryptococcus neoformans dihydrofolate reductase.
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DOI：
10.1016/s0021-9258(18)52956-2
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发表时间：
1993-04
期刊：
影响因子：
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通讯作者：
W. Sirawaraporn;M. Cao;D. Santi;J. Edman
中科院分区：
文献类型：
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作者：
W. Sirawaraporn;M. Cao;D. Santi;J. Edman
新型隐球菌二氢叶酸还原酶 (DHFR) 基因已从 cDNA 和基因组 DNA 文库中分离出来。 690 个碱基对的编码序列编码一个 25,152 Da 的蛋白质，这是迄今为止报道的最大的单功能 DHFR。该基因含有两个内含子，并且已经鉴定了几个假定的调控序列。将编码序列置于基于pUC的表达载体中，该载体在大肠杆菌中以总可溶性提取物的约5%的水平表达新型隐球菌DHFR。通过甲氨蝶呤-琼脂糖亲和层析，然后在 Q-琼脂糖上进行阴离子交换层析，将表达的 DHFR 纯化至均质。在 SDS 聚丙烯酰胺凝胶电泳上，纯化的酶作为单一蛋白质迁移，表观质量为 28 kDa。通过电喷雾质谱分析测定的分子量和氨基末端序列与根据DNA序列预测的一致。几种抗叶酸药物的稳态动力学参数、pH 值、盐和抑制常数的影响已经确定。
The Cryptococcus neoformans dihydrofolate reductase (DHFR) gene has been isolated from cDNA and genomic DNA libraries. The 690-base pair coding sequence codes for a 25,152-Da protein, which is the largest monofunctional DHFR yet reported. The gene contains two introns, and several putative regulatory sequences have been identified. The coding sequence was placed in a pUC-based expression vector, which expresses C. neoformans DHFR in Escherichia coli at a level of about 5% of the total soluble extract. The expressed DHFR was purified to homogeneity by methotrexate-Sepharose affinity chromatography, followed by anion exchange chromatography on Q-Sepharose. On SDS-polyacrylamide gel electrophoresis, the purified enzyme migrates as a single protein with apparent mass of 28 kDa. The molecular weight, as determined by electrospray mass spectral analysis, and the amino-terminal sequence are in accord with what was predicted from the DNA sequence. Steady state kinetic parameters, effects of pH, salts, and inhibition constants of several anti-folates have been determined.