Ruymgaart Arnold P, Heater Sheila J, Oehlers Leon P, Rains J Douglas, Walter Ronald B
Molecular Biosciences Research Group, Department of Chemistry and Biochemistry, Texas State University, San Marcos, Texas 78666, USA.
Zebrafish. 2004;1(3):273-85. doi: 10.1089/zeb.2004.1.273.
The cloning, gene structure, and expression of flap endonuclease-1 (xiFEN1) from Xiphophorus maculates are presented. The xiFEN1 gene structure was found to include 8 exons and 7 introns. The Xiphophorus FEN1 cDNA sequence contained an open reading frame that encoded a 380 amino acid protein with a predicted mass of 43 kDa. The intact FEN1 cDNA was subcloned into a bacterial expression vector (pET101-xiFEN1ct) and recombinant xiFEN1 enzyme purified from E. colicell extracts. The pET101-xiFEN1ct translation product was a 3' fusion protein with a ~3 kDa vector-encoded carboxy terminal extension designed to facilitate protein recognition and purification. The xiFEN1 fusion protein was purified and its amino acid sequence verified by Western blot analysis and tryptic peptide mass fingerprinting. The purified recombinant protein was assessed for enzyme specificity using several different oligonucleotide substrates having select flap overhangs. Also reported are Michaelis steady state kinetic values of enzymatic activity for the xiFEN1 directly compared with human FEN1 activity. xiFEN1 displayed a five-fold greater Km and six-fold lower catalytic efficiency (kcat/Km) than observed for the hFEN1.
本文介绍了剑尾鱼（Xiphophorus maculates）中瓣内切核酸酶-1（xiFEN1）的克隆、基因结构及表达情况。研究发现xiFEN1基因结构包含8个外显子和7个内含子。剑尾鱼FEN1 cDNA序列含有一个开放阅读框，编码一个380个氨基酸的蛋白质，预测分子量为43 kDa。完整的FEN1 cDNA被亚克隆到细菌表达载体（pET101-xiFEN1ct）中，并从大肠杆菌细胞提取物中纯化出重组xiFEN1酶。pET101-xiFEN1ct翻译产物是一种3'融合蛋白，带有一个约3 kDa的载体编码羧基末端延伸，旨在促进蛋白质识别和纯化。通过蛋白质免疫印迹分析和胰蛋白酶肽质量指纹图谱对xiFEN1融合蛋白进行纯化并验证其氨基酸序列。使用几种具有特定瓣状突出端的不同寡核苷酸底物评估纯化的重组蛋白的酶特异性。还报告了直接与人类FEN1活性比较的xiFEN1酶活性的米氏稳态动力学值。与hFEN相比，xiFEN1的Km值高5倍，催化效率（kcat/Km）低6倍。
