A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells |
| |
Authors: | Cazalla Demian Sanford Jeremy R Cáceres Javier F |
| |
Institution: | MRC Human Genetics Unit, Edinburgh, Scotland, UK. |
| |
Abstract: | Here, we describe a simple and efficient method for the expression and purification of active recombinant proteins in mammalian cells. This method uses the expression of T7 epitope-tagged proteins in transiently transfected 293T cells grown in monolayer, followed by anti-T7-agarose affinity chromatography. This procedure yields approximately between 75 and 100 microg of biologically active protein/150 cm(2) flask that can be used for biochemical studies. We have tested this protocol for the expression of the prototype SR protein, SF2/ASF, which is a member of the SR protein family with a role in constitutive and alternative splicing. We show that SF2/ASF purified using this protocol is able to complement an S100 HeLa extract, demonstrating that is biologically active. Moreover, expression of a novel SR-related protein that it is required for the second step of pre-mRNA splicing also rendered an active protein. In summary, we present a protocol based on transient transfection of mammalian cells that results in easy purification of significant amounts of biologically active proteins. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|