Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev.
Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins' application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense.
Marenchino
Marco
M
Medical University of South Carolina, Department of Biochemistry and Molecular Biology, 173 Ashley Avenue, BSB 535D, P.O. Box 250509, Charleston, SC 29425, USA.
Armbruster
David W
DW
Hennig
Mirko
M
D002846:Chromatography, Affinity / Q000379:methods*; D015497:HIV-1 / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D014508:Urea / Q000494:pharmacology; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000302:isolation & purification*
D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D014508:Urea