Wilquet V, Gaspar J A, van de Lande M, Van de Casteele M, Legrain C, Meiering E M, Glansdorff N
Research Institute CERIA-COOVI, Brussels, Belgium.
Eur J Biochem. 1998 Aug 1;255(3):628-37. doi: 10.1046/j.1432-1327.1998.2550628.x.
We have overexpressed the gene for dihydrofolate reductase (DHFR) from Thermotoga maritima in Escherichia coli and characterized the biochemical properties of the recombinant protein. This enzyme is involved in the de novo synthesis of deoxythymidine 5'-phosphate and is critical for cell growth. High levels of T. maritima DHFR in the new expression system conferred resistance to high levels of DHFR inhibitors which inhibit the growth of non-recombinant cells. The enzyme was purified to homogeneity in the following two steps: heat treatment followed by affinity chromatography or cation-exchange chromatography. Most of the biochemical properties of T. maritima DHFR resemble those of other bacterial or eukaryotic DHFRs, however, some are unique to T. maritima DHFR. The pH optima for activity, Km for substrates, and polypeptide chain length of T. maritima DHFR are similar to those of other DHFRs. In addition, the secondary structure of T. maritima DHFR, as measured by circular dichroism, is similar to that of other DHFRs. Interestingly, T. maritima DHFR exhibits some characteristics of eukaryotic DHFRs, such as a basic pI, an excess of positively charged residues in the polypeptide chain and activation of the enzyme by inorganic salts and urea. Unlike most other DHFRs which are monomeric or part of a bifunctional DHFR-thymidylate synthase (TS) enzyme, T. maritima DHFR seems to generally form a dimer in solution and is also much more thermostable than other DHFRs. It may be that dimer formation is a key factor in determining the stability of T. maritima DHFR.
我们已在大肠杆菌中过表达了来自嗜热栖热菌的二氢叶酸还原酶（DHFR）基因，并对重组蛋白的生化特性进行了表征。该酶参与脱氧胸苷5'-磷酸的从头合成，对细胞生长至关重要。新表达系统中高水平的嗜热栖热菌DHFR赋予了对高水平DHFR抑制剂的抗性，这些抑制剂会抑制非重组细胞的生长。该酶通过以下两步纯化至同质：热处理，然后进行亲和色谱或阳离子交换色谱。嗜热栖热菌DHFR的大多数生化特性与其他细菌或真核生物的DHFR相似，然而，有些特性是嗜热栖热菌DHFR所独有的。嗜热栖热菌DHFR的活性最适pH值、底物的Km值和多肽链长度与其他DHFR相似。此外，通过圆二色性测量的嗜热栖热菌DHFR的二级结构与其他DHFR相似。有趣的是，嗜热栖热菌DHFR表现出一些真核生物DHFR的特征，如碱性pI、多肽链中带正电荷残基过多以及被无机盐和尿素激活。与大多数其他单体或双功能DHFR-胸苷酸合酶（TS）酶一部分的DHFR不同，嗜热栖热菌DHFR似乎通常在溶液中形成二聚体，并且也比其他DHFR更耐热。可能二聚体的形成是决定嗜热栖热菌DHFR稳定性的关键因素。
