Pedersen-Lane J, Maley G F, Chu E, Maley F
New York State Department of Health, Wadsworth Center, Albany 12201, USA.
Protein Expr Purif. 1997 Jul;10(2):256-62. doi: 10.1006/prep.1997.0750.
A method is presented for expressing human thymidylate synthase (TS) to the extent of 25-30% of the protein in Escherichia coli. By this procedure, 200-400 mg of pure enzyme can be obtained from a 2-liter culture of cells. The key to the level of expression appears to be related to the conversion of purine bases in the third, fourth, and fifth codons of the TS cDNA to thymine, without altering the encoded protein product. Conversion of the penultimate proline to a leucine did not diminish expression, but while the isolated native enzyme contained only proline on its amino-terminal end, the mutated enzyme was found to contain methionine on its amino terminus. By contrast, the expression of the unmodified TS cDNA represented only about 0.1-0.2% of the total cellular protein. Unlike recombinant rat and human TSs, the respective enzymes purified to homogeneity from eukaryotic cells were blocked at the amino ends and possessed 2- to 4-fold lower specific activities. To determine at what level the impairment of expression occurred, an in vitro transcription, translation system was employed and the results showed that while transcription was unaffected, the translation of native TS mRNA was reduced by at least 20-fold relative to modified TS mRNA using a rabbit reticulocyte translation system. Thus, it appears that at least for the TS gene, expression is greatly influenced by the GC content of the 5' coding region of the gene in both prokaryote and eukaryote systems.
本文介绍了一种在大肠杆菌中表达人胸苷酸合成酶（TS）的方法，其表达量可达细胞总蛋白的25%-30%。通过该方法，从2升细胞培养物中可获得200-400毫克纯酶。表达水平的关键似乎与将TS cDNA第三、第四和第五密码子中的嘌呤碱基转换为胸腺嘧啶有关，而不改变编码的蛋白质产物。将倒数第二个脯氨酸转换为亮氨酸不会降低表达，但分离出的天然酶在其氨基末端仅含有脯氨酸，而突变酶在其氨基末端含有甲硫氨酸。相比之下，未修饰的TS cDNA的表达仅占细胞总蛋白的约0.1%-0.2%。与重组大鼠和人TS不同，从真核细胞中纯化至同质的相应酶在氨基末端被阻断，比活性低2-4倍。为了确定表达受损发生在哪个水平，采用了体外转录、翻译系统，结果表明，虽然转录不受影响，但使用兔网织红细胞翻译系统时，天然TS mRNA的翻译相对于修饰的TS mRNA至少降低了20倍。因此，似乎至少对于TS基因，在原核生物和真核生物系统中，基因5'编码区的GC含量对表达有很大影响。
