Heterologous overexpression and purification of four common subunits of nuclear RNA polymerases I, II and III of Schizosaccharomyces pombe.
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.
Proshkin
Sergey A
SA
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP-7, 117997, Moscow, Russia.
Shpakovski
George V
GV
D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003035:Cobalt / Q000737:chemistry; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D009532:Nickel / Q000737:chemistry; D012318:RNA Polymerase I / Q000096:biosynthesis* / Q000302:isolation & purification*; D012319:RNA Polymerase II / Q000096:biosynthesis* / Q000302:isolation & purification*; D012320:RNA Polymerase III / Q000096:biosynthesis* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003035:Cobalt; D009532:Nickel; D012319:RNA Polymerase II; D012318:RNA Polymerase I; D012320:RNA Polymerase III