Purification of the growth hormone releasing hormone receptor with a C-terminal, biotinylated affinity ligand.
Purification of the growth hormone releasing hormone receptor with a C-terminal, biotinylated affinity ligand.
复制标题
使用 C 端生物素化亲和配体纯化生长激素释放激素受体。
DOI：
10.1006/bbrc.1996.0540
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发表时间：
1996
影响因子：
3.1
通讯作者：
Thorner,MO
中科院分区：
文献类型：
--
作者：
Zysk,JR;Gaylinn,BD;Lyons,CE;Johnson,B;Eppler,CM;Baumbach,WR;Thorner,MO
利用生物素化的生长激素释放激素类似物，从牛脑垂体组织和转染人或猪受体的HEK293细胞中纯化了生长激素释放激素受体(GHRH)。定制合成的[His1，Nle27，Biotin-Lys41]-人GHRH-(1-41)-NH2(GHRHb)与脑垂体膜结合，亲和力与人GHRH相当。GHRHb在多肽的C端具有生物素基团，可以同时与受体和链霉亲和素琼脂糖结合。该类似物直接用于从垂体组织中纯化GHRH受体，或通过掺入光亲和基团ANBNOSb(Ghrhλb)进行修饰，放射性碘标记并用于从转染的HEK293细胞膜中纯化GhrH受体。制备膜并与各自的配体预结合，然后进行CHAPS增溶，并将增溶的络合物应用于链霉亲和素琼脂糖柱。用银染的十二烷基硫酸钠凝胶电泳法或HEK293洗脱液的凝胶放射自显影分析，分别显示52和55 kDa的特异条带。对于配体交联型受体，预期后一条带的大小更大。用N-糖苷酶处理后，这两条带显示了相似的10 kDa的迁移率移动，这是一种以前用于表征这种受体的方法(1)。在银染凝胶的洗脱液中也检测到一条与GSα亚基大小相对应的45 kDa条带，表明GHRH受体是以异源三聚体的形式被回收的。该方法的折叠纯度和产率分别大于5万和2.6-9%。
The receptor for growth hormone-releasing hormone (GHRH) has been purified from bovine pituitary tissue and HEK293 cells transfected with human or porcine receptor using a retrievable biotinylated GHRH analog. Custom synthesized [His1, Nle27, Biotin-Lys41]-human GHRH-(1–41)-NH2(GHRHb) bound to pituitary membranes with affinity comparable to human GHRH. GHRHbwhich has the biotinyl group on the C-terminus of the peptide allowed simultaneous binding to both the receptor and streptavidin agarose. This analog was used directly in the purification of the receptor from pituitary tissue or was modified by incorporation of the photoaffinity group ANBNOS (GHRHλb), radioiodinated and used to demonstrate purification of the GHRH receptor from transfected HEK293 cell membranes. Membranes were prepared and prebound with the respective ligand followed by CHAPS-solubilization and application of the solubilized complex to a strepavidin agarose column. Analysis of eluates from the pituitary tissue purification by silver stained SDS PAGE or of autoradiographs of gels from HEK293 eluates revealed specific bands of 52 and 55 kDa, respectively. The higher size of the latter band is expected for the ligand-crosslinked receptor. Both bands displayed similar mobility shifts of 10 kDa upon treatment with N-glycosidase, a method previously used to characterize this receptor (1). A 45 kDa band corresponding to the size of the GSαsubunit was also detected in eluates of the silver stained gels, suggesting that the GHRH receptor was retrieved as a heterotrimeric complex. Fold purification and yield for this procedure were estimated to be greater than 50,000 and 2.6–9%, respectively.