血红素加氧酶在大肠杆菌中稳定表达的氨基酸范围的研究  

Research of Amino Acid Range of Heme Oxygenase Allowing Stable Expression as Soluble Proteins in E. coli

作　　者：王秀宏[1] 滕悦秋[2] 王志刚[2] 刘明[1] 周虹[2] 

机构地区：[1]哈尔滨医科大学生物化学与分子生物学教研室,哈尔滨市150086 [2]军事医学科学院野战输血研究所,北京市100850

出　　处：《医学分子生物学杂志》2004年第4期212-215,共4页Journal of Medical Molecular Biology

基　　金：国家自然科学基金 (No 3 0 0 0 0 0 3 0 ) ;黑龙江省杰出青年基金 ( 2 0 0 1)

摘　　要：目的　确定在原核细胞中呈可溶性稳定表达但不改变活性的血红素加氧酶氨基酸范围。方法　应用PCR技术构建了鼠血红素加氧酶 1(RHO 1)和人血红素加氧酶 2 (HHO 2 )的N端和C端部分氨基酸缺失的变异型RHO 1和HHO 2 ,将它们在大肠杆菌中表达并纯化后通过光谱扫描法测定变异型酶与野生型酶的催化活性 ,确定RHO 1和HHO 2中的N端和C端缺失不影响其可溶性表达及催化活性的氨基酸范围。结果　△N8RHO 1(N端缺失 8个氨基酸的RHO 1)和△C70RHO 1在大肠杆菌中表达为可溶性蛋白且催化活性与RHO 1野生型相同 ,△N2 0HHO 2和△C77HHO 2在大肠杆菌中也表达为可溶性蛋白且催化活性与HHO 2野生型相同 ,而△N9RHO 1和△C75RHO 1在大肠杆菌中表达为包涵体。结论　RHO 1和HHO 2的N端和C端部分氨基酸缺失的突变型酶对其催化功能没有影响 ,并可在大肠杆菌中稳定可溶性表达。Objective In order to define the range of critical amino acids of heme oxygenase, which is expressed stably as soluble proteins in prokaryotic cells. Methods We established the deletion mutants of rat heme oxygenase-1 (RHO-1) and human heme oxygenase-2 (HHO-2) successfully by PCR. They were expressed in Escherichia coli (E. coli), purified and estimated catalytic activity by spectrum scan assay.Results We confirmed the range of amino acids under which N-terminal and c-terminal amino acid sequences in RHO-1 and HHO-2 had no effect on their stable expression and catalytic function by comparing catalytic activity between mutant and wild enzyme. There were no significant differences between deleting 8-amino acid N-terminal and 70-amino acid C-terminal mutants of RHO-1 in terms of soluble proteins in E. coli with wild RHO-1. No significant differences were found between deleting 20-amino acid N-terminal and 77-amino acid C-terminal mutants of HHO-2 in terms of soluble proteins expressed in E. coli with wild HHO-2, but deleting 9-amino acid N-terminal and 75-amino acid C-terminal mutants of RHO-1 were expressed as inclusion bodies in E. coli. Conclusion Our study demonstrated that RHO-1and HHO-2 deleting partial N-terminal and C-terminal amino acid sequences have no effect on their catalytic function, and they are stably expressed as soluble proteins in E. coli.

关 键 词：血红素加氧酶 大肠杆菌 缺失的变异型 催化活性 

分 类 号：Q55[生物学—生物化学]