Cloning, overexpression, purification, and physicochemical characterization of a cold shock protein homolog from the hyperthermophilic bacterium Thermotoga maritima
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Media type:
E-Article
Title:
Cloning, overexpression, purification, and physicochemical characterization of a cold shock protein homolog from the hyperthermophilic bacterium Thermotoga maritima
Description:
<jats:title>Abstract</jats:title><jats:p><jats:italic>Thermotoga maritima (Tm</jats:italic>) expresses a 7 kDa monomeric protein whose 18 N‐terminal amino acids show 81% identity to N‐terminal sequences of cold shock proteins (Csps) from <jats:italic>Bacillus caldolyticus</jats:italic> and <jats:italic>Bacillus stearothermophilus</jats:italic>. There were only trace amounts of the protein in <jats:italic>Thermotoga</jats:italic> cells grown at 80°C. Therefore, to perform physicochemical experiments, the gene was cloned in <jats:italic>Escherichia coli</jats:italic>. A DNA probe was produced by PCR from genomic <jats:italic>Tm</jats:italic> DNA with degenerated primers developed from the known N‐terminus of <jats:italic>Tm</jats:italic>Csp and the known C‐terminus of CspB from <jats:italic>Bacillus subtilis</jats:italic>. Southern blot analysis of genomic <jats:italic>Tm</jats:italic> DNA allowed to produce a partial gene library, which was used as a template for PCRs with gene‐and vector‐specific primers to identify the complete DNA sequence. As reported for other <jats:italic>csp</jats:italic> genes, the 5′ untranslated region of the mRNA was anomalously long; it contained the putative Shine–Dalgarno sequence. The coding part of the gene contained 198 bp, i.e., 66 amino acids. The sequence showed 61% identity to CspB from <jats:italic>B. caldolyticus</jats:italic> and high similarity to all other known Csps. Computer‐based homology modeling allowed the conclusion that <jats:italic>Tm</jats:italic>Csp represents a β‐barrel similar to CspB from <jats:italic>B. subtilis</jats:italic> and CspA from <jats:italic>E. coli</jats:italic>.</jats:p><jats:p>As indicated by spectroscopic analysis, analytical gel permeation chromatography, and mass spectrometry, over‐expression of the recombinant protein yielded authentic <jats:italic>Tm</jats:italic>Csp with a molecular weight of 7,474 Da. This was in agreement with the results of analytical ultracentrifugation confirming the monomeric state of the protein. The temperature‐induced equilibrium transition at 87 °C exceeds the maximum growth temperature of <jats:italic>Tm</jats:italic> and represents the maximal <jats:italic>T<jats:sub>m</jats:sub></jats:italic>‐value reported for Csps so far.</jats:p>