A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells.
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.
Cazalla
Demian
D
MRC Human Genetics Unit, Edinburgh, Scotland, UK.
Sanford
Jeremy R
JR
Cáceres
Javier F
JF
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D000906:Antibodies / Q000276:immunology; D017123:Bacteriophage T7 / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002846:Chromatography, Affinity; D015870:Gene Expression / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors
D000906:Antibodies; D036022:Capsid Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000068103:Serine-Arginine Splicing Factors