Murray Samuel, Maraveyas Anthony, Dougan Tammy, Chu Anthony C
Unit of Dermatology, Department of Medicine, ICSTM, Hammersmith Campus, London W12 0HS, UK.
Biochim Biophys Acta. 2002 Mar 15;1570(2):81-8. doi: 10.1016/s0304-4165(02)00157-5.
Naturally occurring core-Streptavidin (c-Strep) would serve as a more useful agent in vivo if not for its high kidney retention. This retention is mediated by an integrin-binding motif-RYDS-that shares homology to the more common RGDS. We generated a c-Strep molecule constituting amino acids 13-139 of streptavidin and by site-directed mutagenesis altered the RYDS motif to RYES. RYDS-c-Streptavidin and RYES-c-Streptavidin were expressed in E. coli and purified on a 2-imminobiotin matrix. Each demonstrated an affinity for biotin similar to that of native post-secretory streptavidin while maintaining their ability to form dimers and tetramers. The mutant RYES-c-Streptavidin was no longer able to mediate normal rat kidney cell attachment in an in vitro assay. RYDS-c-Streptavidin-mediated kidney cell attachment was inhibited by competition with c-Streptavidin, RYDS-c-Streptavidin and RGDS-containing peptides but not with an irrelevant peptide or RYES-c-Streptavidin. Therefore, the point mutation D49E generates a molecule, which may not display the in vivo kidney retention observed for RYDS-c-Streptavidin, potentially finding more widespread clinical application.
天然存在的核心链霉亲和素（c-Strep）若不是肾脏潴留率高，在体内会是一种更有用的药物。这种潴留是由一个整合素结合基序-RYDS-介导的，它与更常见的RGDS具有同源性。我们生成了一个由链霉亲和素的13-139位氨基酸组成的c-Strep分子，并通过定点诱变将RYDS基序改变为RYES。RYDS-c-链霉亲和素和RYES-c-链霉亲和素在大肠杆菌中表达，并在2-亚氨基生物素基质上纯化。每一种都表现出与天然分泌后链霉亲和素相似的对生物素的亲和力，同时保持其形成二聚体和四聚体的能力。在体外试验中，突变体RYES-c-链霉亲和素不再能够介导正常大鼠肾细胞附着。与c-链霉亲和素、RYDS-c-链霉亲和素和含RGDS的肽竞争可抑制RYDS-c-链霉亲和素介导的肾细胞附着，但与无关肽或RYES-c-链霉亲和素竞争则不能。因此，D49E点突变产生了一种分子，它可能不会表现出RYDS-c-链霉亲和素在体内观察到的肾脏潴留现象，可能会有更广泛的临床应用。
