蜕皮甾体受体的磁性DNA亲和纯化

Magnetic DNA affinity purification of ecdysteroid receptor.

作者信息

Ozyhar A, Gries M, Kiltz H H, Pongs O

机构信息

Ruhr-Universität Bochum, Lehrstuhl für Biochemie, Bochum, Germany.

出版信息

J Steroid Biochem Mol Biol. 1992 Dec;43(7):629-34. doi: 10.1016/0960-0760(92)90287-s.

DOI:10.1016/0960-0760(92)90287-s

PMID:1472454

Abstract

A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity. The second step involved the use of a magnetic DNA affinity technique where the double stranded hsp 27 oligonucleotide containing EcdR binding sequence was biotin 5'-end labelled and bound to monodisperse superparamagnetic particles coated with streptavidin (Dynabeads M-280 Streptavidin) giving magnetic DNA affinity beads. The chromatographed EcdR-ponasterone A complexes were bound to the magnetic DNA affinity beads and by magnetic separation, wash and elution, a 29,000-fold enriched EcdR preparation was obtained within 1.5 h. This procedure can be applied for other EcdR sources with minor modifications.

摘要

本文介绍了一种从黑腹果蝇核提取物中快速纯化蜕皮甾体受体（EcdR）至近乎同质状态的新方法。在纯化过程的第一步中，用[3H]蜕皮甾酮A对EcdR分子进行放射性标记，然后在非常温和的条件下，将[3H]蜕皮甾酮A-EcdR复合物在Fractogel EMD TMAE（s）离子交换剂上进行色谱分离。得到了纯化23倍的受体，其可保存在液氮中而不失活。第二步采用磁珠DNA亲和技术，将含有EcdR结合序列的双链hsp 27寡核苷酸在5'-末端用生物素标记，并与包被有链霉亲和素的单分散超顺磁性颗粒（Dynabeads M-280链霉亲和素）结合，制成磁珠DNA亲和珠。将经色谱分离的EcdR-蜕皮甾酮A复合物与磁珠DNA亲和珠结合，通过磁分离、洗涤和洗脱，在1.5小时内获得了富集29000倍的EcdR制剂。该方法稍作修改即可应用于其他EcdR来源。