| 黑曲霉硫氧还蛋白的克隆表达纯化及其酶活位点特征分析 |
| Gene cloning, expression and characterization of a thioredoxin from Aspergillus niger |
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| 中文关键词:定点突变，催化活性，比浊法 |
| 英文关键词:site-directed mutation, catalytic activity, turbidimetry |
| 基金项目:国家自然科学基金重点项目（No. 30930018）和国家“973计划”（No. 2009CB118904） |
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| 中文摘要: |
| 首次从黑曲霉Aspergillus niger全基因组中克隆出黑曲霉硫氧还原蛋白基因AnTrx，并对其编码蛋白的第33－37位保守区的活性位点实施定点突变C34S、C37S 及C34S-C37S，获得相应的3个定点突变基因。将野生型AnTrx及其突变子分别在大肠杆菌Escherichia coli中诱导表达，比浊法测定纯化的各表达产物还原牛胰岛素a与b链之间二硫键的活性。结果表明，AnTrx的3个突变体都不表现明显催化活性。当突变型与野生型AnTrx等量混合后，发现突变型AnTrx-C34S可显著提高野生型AnTrx的催化效率，而突变型AnTrx-C37S却无此功能。由此证明，AnTrx活性结构域的第37位Cys残基上的巯基能参与攻击硫氧还蛋白和底物蛋白所形成的二硫键而释放被还原的底物蛋白，而第34位Cys残基同其他微生物的同一活性域一样参与硫氧化还蛋白与底物的结合。这一结果有助于认识真菌硫氧还蛋白第37位活性位点的作用。 |
| 英文摘要: |
| A gene encoding Aspergillus niger thioredoxin (AnTrx) was cloned for the first time from the fungal genome. Three site-directed mutations (C34S, C37S and C34S-C37S) of the cloned gene AnTrx generated the mutants AnTrx-C34S, AnTrx-C37S and AnTrx-C34S-C37S. The wild and mutated genes were separately transformed into Escherichia coli for expression. As a result, the three mutated proteins purified from Escherichia coli cultures showed no substantial activity in the reduction of insulin compared to the wild-type AnTrx. Interestingly, the mutant AnTrx-C34S significantly enhanced the activity of the purified AnTrx when both proteins were added to the reaction system in equal volume but there was no difference in activity between AnTrx and AnTrx-C37S. Our results indicate that the residue Cys at AnTrx site 37 may participate in the second step of the reduction reaction, which generates reduced protein and disulfide-vectoring thioredoxin, while the same residue at AnTrx site 34 is involved only in the first step of the reaction as usual in microbial thioredoxins. |
| 王晓慧,应盛华,冯明光. 黑曲霉硫氧还蛋白的克隆表达纯化及其酶活位点特征分析[J]. 菌物学报, 2010, 29(3): 409-413 |
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