title: Cloning, Escherichia coli expression, purification, characterization, and enzyme assay of the ribosomal protein S4 from wheat seedlings (Triticum vulgare).
abstract: S4 is a paradigm of ribosomal proteins involved in multifarious activities both within and outside the ribosome. For a detailed biochemical and structural investigations of eukaryotic S4, the wheat S4 gene has been cloned and expressed in Escherichia coli, and the protein purified to a high degree of homogeneity. The 285-residue recombinant protein containing an N-terminal His(6) tag along with fourteen additional residues derived from the cloning vector is characterized by a molecular mass of 31981.24 Da. The actual sequence of 265 amino acids having a molecular mass of 29931 Da completely defines the primary structure of wheat S4. Homology modeling shows a bi-lobed protein topology arising from folding of the polypeptide into two domains, consistent with the fold topology of prokaryotic S4. The purified protein is stable and folded since it can be reversibly unfolded in guanidinium hydrochloride, and is capable of hydrolyzing cysteine protease-specific peptide-based fluorescence substrates, including Ac-DEVD-AFC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin).
authors.affiliation: School of Chemistry, University of Hyderabad, Hyderabad 500 046, India.
authors.forename: Madasu
authors.initials: M
authors.lastname: Yadaiah
authors.affiliation: School of Chemistry, University of Hyderabad, Hyderabad 500 046, India.
authors.forename: P
authors.initials: P
authors.lastname: Nageswara Rao
authors.affiliation: School of Chemistry, University of Hyderabad, Hyderabad 500 046, India.
authors.forename: Babu
authors.initials: B
authors.lastname: Sudhamalla
authors.affiliation: School of Chemistry, University of Hyderabad, Hyderabad 500 046, India.
authors.forename: Dasari
authors.initials: D
authors.lastname: Ramakrishna
authors.affiliation: School of Chemistry, University of Hyderabad, Hyderabad 500 046, India.
authors.forename: U
authors.initials: U
authors.lastname: Mahammad Yasin
authors.affiliation: School of Chemistry, University of Hyderabad, Hyderabad 500 046, India; School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India. Electronic address: akbsc@uohyd.ernet.in.
authors.forename: Abani K
authors.initials: AK
authors.lastname: Bhuyan
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D019791:Guanidine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D058767:Protein Unfolding; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D036226:Seedlings / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D053719:Tandem Mass Spectrometry; D014908:Triticum / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D003374:Coumarins; D017931:DNA Primers; D009842:Oligopeptides; D012269:Ribosomal Proteins; C031798:ribosomal protein S4; D019791:Guanidine