用单价STV制备单分子蛋白质-DNA复合物的研究

摘要

Monovalent streptavidin(STV)was prepared through renaturation of denatured mutant with inactivated biotin binding sites and wild-type STV mixture by a molar ratio of 3 ∶ 1,followed by purification through a nickel affinity column as previously reported.Biotinylated DNA of 500 bp was incubated with monovavent STV at different molar ratios to screen the best ratio for preparation of 1STV-1DNA single-molecule conjugates.Results showed that,among the molar ratio gradients selected in this study,1 ∶ 1 ratio of STV to DNA was optimal.This single protein-DNA conjugate method using monovalent STV was significantly superior to that using wild-type STV,providing a convenient and highly efficient approach for applications in the study of DNA manipulation,DNA-protein interaction and fabrication of DNA chips at the single molecule level.%将生物素结合位点失活的链霉亲和素(streptavidin,STV)变异体变性后的亚基与野生型STV变性后的亚基以摩尔比3∶1的比例混合,然后重折叠复性,镍柱层析分离得到只有一个有活性生物素结合位点的突变体STV,即单价STV.将单价STV与500 bp一端生物素标记的双链DNA(biotin-DNA)按不同摩尔比混合孵育,探讨使用单价STV制备1STV-1DNA复合物的可行性,并筛选制备1STV-1DNA复合物的最佳混合摩尔比.结果表明,在所用的摩尔比梯度中,单价STV与biotin-DNA混合的最佳摩尔比为1∶1,与使用野生型STV制备方法相比,使用单价STV更为简便、高效,为单分子DNA操纵、单分子DNA-蛋白质相互作用研究以及单分子DNA芯片的制备等提供了便捷的方法.