嗜热菌铁硫簇合成途径中SufB、SufC和SufD蛋白的表达、纯化及其功能初步研究

摘要

铁硫簇广泛存在于各种生物中,参与电子传递、底物结合和激活、基因表达和调控等重要功能.本文利用嗜热菌Thermus thermophilus HB8研究铁硫簇合成SUF系统中的三个关键蛋白SufB、SufC、SufD.以HB8的基因组为模板,通过PCR技术获得其su、sufC、sufD三个基因,随后构建了蛋白表达载体sufB-pET28a(+)、sufC-pET28a(+)、sufD-pGEX-6P-1,将其转入大肠杆菌Rosetta( DE3)进行蛋白表达,再利用亲和层析与凝胶过滤层析进行蛋白纯化.随后对SufB、SufC、SufD功能进行了初步研究,证明SufC对ATP具有亲和性,其亲和常数为15.2 μmol/L；证明SufB与SufC在体外具有相互作用,并得到了复合体SufBC,为进一步探讨其作用机制奠定了基础.%Iron-sulfur cluster,ubiquitous in all living organisms, is one of the oldest living matter. In this paper, sufB, sufC,sufD were cloned by PCR using HB8 genome as a template. Three protein expression vectors, su/Z?-pET28a ( + ) , sufC-pET28a ( + ) , sjf/D-pGEX-6P-l were constructed and transferred into Rosetta ( DE3 ) for protein expression. Proteins of Thermus thermophilic HB8 have thermal stability, which is conducive to reconstructing the complex and some preliminary functional studies. Further research on the mechanism is continued and processing.