Large-scale overexpression and purification of ADARs from Saccharomyces cerevisiae for biophysical and biochemical studies.
Many biochemical and biophysical analyses of enzymes require quantities of protein that are difficult to obtain from expression in an endogenous system. To further complicate matters, native adenosine deaminases that act on RNA (ADARs) are expressed at very low levels, and overexpression of active protein has been unsuccessful in common bacterial systems. Here we describe the plasmid construction, expression, and purification procedures for ADARs overexpressed in the yeast Saccharomyces cerevisiae. ADAR expression is controlled by the Gal promoter, which allows for rapid induction of transcription when the yeast are grown in media containing galactose. The ADAR is translated with an N-terminal histidine tag that is cleaved by the tobacco etch virus protease, generating one nonnative glycine residue at the N-terminus of the ADAR protein. ADARs expressed using this system can be purified to homogeneity, are highly active in deaminating RNA, and are produced in quantities (from 3 to 10mg of pure protein per liter of yeast culture) that are sufficient for most biophysical studies.
Department of Biochemistry and Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah, USA.
Mark R
MR
Macbeth
Brenda L
BL
Bass
D000243:Adenosine Deaminase / Q000737:chemistry* / Q000302:isolation & purification*; D001483:Base Sequence; D001671:Biochemistry / Q000379:methods*; D001703:Biophysics / Q000379:methods*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005998:Glycine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D016601:RNA-Binding Proteins; D012313:RNA; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase; D005998:Glycine