Abstract
Four subunits of Schizosaccharomyces pombe RNA polymerases I-III shared by all three enzymes (Rpb5, Rpb8, Rpb10 and Rpc10 [Rpb12]) have been overexpressed in Escherichia coli expression vectors pQE or pET as hexahistidine fusions. The recombinant proteins have been purified to near homogeneity using metal-chelate affinity chromatography and gel filtration. Homogeneity and identity of the purified protein preparations was demonstrated by denaturing polyacrylamide gel electrophoresis and TOF-MALDI mass spectrometry. The proteins were obtained in large amounts, and their preparations are currently in use for monoclonal antibody production and physico-chemical studies of these individual components of eukaryotic transcription enzymes.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Nucleus / chemistry
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Cell Nucleus / metabolism
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Chemical Phenomena
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Chemistry, Physical
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Chromatography, Affinity
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Chromatography, Gel
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Cloning, Molecular
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Cobalt / chemistry
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DNA, Complementary / biosynthesis
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DNA, Complementary / genetics
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression Regulation, Enzymologic
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Gene Expression Regulation, Fungal
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Genetic Vectors
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Nickel / chemistry
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RNA Polymerase I / biosynthesis*
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RNA Polymerase I / isolation & purification*
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RNA Polymerase II / biosynthesis*
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RNA Polymerase II / isolation & purification*
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RNA Polymerase III / biosynthesis*
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RNA Polymerase III / isolation & purification*
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / isolation & purification
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Schizosaccharomyces / enzymology*
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Schizosaccharomyces / metabolism*
Substances
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DNA, Complementary
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Recombinant Proteins
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Cobalt
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Nickel
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RNA Polymerase II
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RNA Polymerase I
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RNA Polymerase III