人类神经型一氧化氮合酶原核表达载体的构建和在大肠杆菌中的表达(英文)

Construction of human neuronal nitric oxide synthase expression system in Escherichia coli

目的 :构建在大肠杆菌中表达重组人神经型一氧化氮合酶的高表达系统。方法 :用PCR方法从cD NA中扩增目的编码基因 ,然后将编码基因连接入表达载体pCWori+,将重组的质粒转染入大肠杆菌BL2 1进行蛋白高表达 ,经Westernblot确认表达后 ,进行大量培养 ,通过层析方法精制此酶 ,并用吸收光谱法对酶的性质进行测定。结果 :从该表达系统中可以获得 3mg/L培养基的高产量的一氧化氮合酶。结论 :从该表达系统中可获得大量有活性的人一氧化氮合酶。

AIM: To construct a high-level expression system of recombinant human neuronal nitric oxide synthase (hnNOS) full-length enzyme in Escherichia coli. METHODS: The coding sequence of hnNOS full-length was firstly amplified by PCR, and then ligated into the expression vector pCWori+. The recombinant plasmid was transformed into Escherichia coli BL21 for high-level expression. After having been checked with Western blot, the enzyme was used for large-scale culture and purification. Finally, the property of the enzyme was determined by spectrophotometric method. RESULTS: The constructed expression system could give a yielding of 3 mg/L initial culture. CONCLUSION: The expression system constructed is fully sufficient to express the active human neuronal nitric oxide synthase.