摘要
铁氧还蛋白ＮＡＤＰ＋氧还酶（ＦＮＲ）是光合电子传递中的关键酶之一。蓝细菌Ｓｙｎｅｃｈｏｃｏｃｕｓｓｐ．ＰＣＣ７００２中编码ＦＮＲ的ｐｅｔＨ基因被克隆到表达载体ｐＥＴ３ｄ上，在大肠杆菌中得到了高效表达，其表达量占大肠杆菌总蛋白的５０％以上。高效表达的产物以可溶性和包含体两种形式存在。可溶性部分具有ＦＮＲ的酶活性，在经过离子交换和凝胶层析后产生了电泳纯的ＦＮＲ。包含体形式的部分经６７ｍｏｌ／Ｌ尿素变性后，可在有黄素腺嘌呤二核苷酸（ＦＡＤ）存在条件下重新折叠成有活性的ＦＮＲ。这两种方式的ＦＮＲ其吸收光谱与从蓝细菌分离的ＦＮＲ的相同，吸收峰位置在２７３ｎｍ、３８５ｎｍ和４５６ｎｍ。氨基末端氨基酸序列分析表明此表达产物确为ｐｅｔＨ所编码。高效表达的ＦＮＲ能催化从Ｐ７００到ＮＡＤＰ＋的电子传递。其黄递酶的酶活性最适ｐＨ为８０，最适温度为３０℃。
Fd:NADP + oxidoreductase (FNR) is one of the key enzymes in photosynthetic electron transport. The gene petH encoding FNR of Synechococcus sp. PCC 7002 was cloned into the expressing vector pET 3 d′ and overexpressed in E.coli. The amount of recombinant FNR (rFNR) was over 50% of the total cellular proteins. There were two forms of FNR activity, one is soluble and the other one was in the form of inclusion bodies. The soluble rFNR was purified through ion exchange chromatography and gel chromatography. The rFNR in the form of inclusion bodies was first solubilized with 6.7 mol/L urea, and then refolded into the active form in the presence of flavin adenine dinucleotide (FAD). Further purification was performed by ion exchange chromatography. The rFNR pruified from either form of the expressed product had the maximum absorption spectrum as that of the natural FNR from cyanobacteria, whose maximum absorption was at 273, 385 and 456 nm respectively. N terminal sequencing showed that rFNR was indeed a product of petH gene expression. rFNR could catalyze the electron transport from P 700 to NADP + in the presence of ferredoxin . The optimal pH for diaphorase activity of rFNR was 8.0 and the optimal temperature was 30 ℃.
基金
国家自然科学基金委
国家教委优秀青年教师基金
