Expression, purification and characterization of individual bromodomains from human Polybromo-1.
Computational analysis reveals six tandem bromodomains within the amino-terminal region of the human Polybromo-1 protein, a required subunit of the Polybromo, BRG1-associated factors chromatin remodeling complex. Bromodomains are acetyl-lysine binding modules found in many chromatin binding proteins and histone acetyltransferases. Recent in vivo studies suggest that bromodomains can both discriminate the presence of an acetyl group on a lysine side chain and locate the acetyl-lysine within a histone protein. Together, this implies that multiple bromodomains may be able to function cooperatively and recognize a specific acetylation pattern to localize remodeling complexes to specific chromatin sites. Here, the cloning, expression and bioactivity of recombinant bromodomains from the human Polybromo-1 protein is described. Individual bromodomains from Polybromo-1 were cloned from human cDNA into a pET30b expression vector enabling effective one-step purification by affinity chromatography. Due to complications, including the high number of rare codons found in the coding regions and the tendency of individually expressed domains to aggregate and misfold, bacterial expression was only achieved using a cell strain containing rare eukaryotic tRNAs. Fluorescence-based bioactivity assays were performed to determine if native binding features were retained. The present cloning, expression, and purification procedure enabled the preparation of large quantity and high yields of biologically active recombinant bromodomains from human Polybromo-1 for in vitro structure and function studies. This is the first report of recombinant active form of bromodomains obtained from PB1.
Chandrasekaran
Renu
R
Department of Chemistry, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA.
Thompson
Martin
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014157:Transcription Factors / Q000235:genetics*
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C102195:PBRM1 protein, human; D011994:Recombinant Proteins; D014157:Transcription Factors; D008239:Lysine