Sugita T, Aya H, Ueno M, Ishizuka T, Kawashima K
Lead Generation Research Laboratory, Tanabe Seiyaku Co., Ltd., Osaka.
J Biochem. 1997 Aug;122(2):309-13. doi: 10.1093/oxfordjournals.jbchem.a021754.
The cDNA encoding human 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase has been cloned from a placenta cDNA library, utilizing a PCR-derived probe. It encodes a peptide of 592 amino acids. The amino (N)-terminal sequence of this enzyme, purified from HeLa cells and CCRF-CEM cells, was found to be APGQLALF-. Both sequencing results revealed a difference of six N-terminal residues when compared to the reported sequence of cloned cDNA from a hepatoma cDNA library. Northern-blot analysis of human AICAR transformylase mRNA showed the expression of a single 2.0 kb mRNA in all tissues examined. With the cloned cDNA fragment, we constructed expression vectors for mature and GST-fused AICAR transformylase. Both recombinant molecules possessing AICAR transformylase activity were overproduced in Escherichia coli. GST-AICAR transformylase can be purified to homogeneity by a single-step affinity procedure with glutathione Sepharose. Mutational analysis, utilizing this expression system, showed that His213 and His267 were essential for AICAR transformylase activity.
利用聚合酶链式反应（PCR）衍生的探针，从胎盘cDNA文库中克隆出了编码人5-氨基咪唑-4-甲酰胺核糖核苷酸（AICAR）转甲酰基酶的cDNA。它编码一个由592个氨基酸组成的肽段。从HeLa细胞和CCRF-CEM细胞中纯化得到的这种酶的氨基（N）末端序列为APGQLALF-。与从肝癌cDNA文库中克隆的cDNA的报道序列相比，这两个测序结果都显示N末端有六个残基的差异。对人AICAR转甲酰基酶mRNA的Northern印迹分析表明，在所检测的所有组织中都有一个单一的2.0 kb mRNA表达。利用克隆的cDNA片段，我们构建了成熟型和GST融合型AICAR转甲酰基酶的表达载体。两种具有AICAR转甲酰基酶活性的重组分子在大肠杆菌中都得到了过量表达。GST-AICAR转甲酰基酶可以通过用谷胱甘肽琼脂糖进行单步亲和纯化程序纯化至同质。利用这个表达系统进行的突变分析表明，His213和His267对AICAR转甲酰基酶活性至关重要。
