Cloning, Escherichia coli expression, purification, characterization, and enzyme assay of the ribosomal protein S4 from wheat seedlings (Triticum vulgare)

• Published in: Protein expression and purification 2012-01, Vol.81 (1), p.55-62
• Main Authors: Yadaiah, Madasu, Nageswara Rao, P., Sudhamalla, Babu, Ramakrishna, Dasari, Mahammad Yasin, U., Bhuyan, Abani K.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Cloning, Molecular; Coumarins - metabolism; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Guanidine; Models, Molecular; Molecular Sequence Data; Oligopeptides - metabolism; Protein Unfolding; Recombinant S4; Ribosomal protein S4; Ribosomal proteins; Ribosomal Proteins - chemistry; Ribosomal Proteins - genetics; Ribosomal Proteins - isolation & purification; Ribosomal Proteins - metabolism; S4 enzyme; Seedlings - chemistry; Seedlings - metabolism; Sequence Alignment; Spectrometry, Fluorescence; Tandem Mass Spectrometry; Triticum - chemistry; Triticum - genetics; Triticum - metabolism; Triticum aestivum; Triticum vulgare
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2011.09.003

► We describe production of wheat ribosomal protein S4 in Escherichia coli. ► The 265-amino acid protein is a hitherto unknown cysteine protease. ► It hydrolyzes cysteine protease-specific peptide-based substrates. ► The proteolytic nature could be related to extraribosomal function of S4. 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 His6 tag along with fourteen additional residues derived from the cloning vector is characterized by a molecular mass of 31981.24Da. The actual sequence of 265 amino acids having a molecular mass of 29931Da 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).