Cloning, overexpression, purification, and physicochemical characterization of a cold shock protein homolog from the hyperthermophilic bacterium Thermotoga maritima

Abstract

Thermotoga maritima (Tm ) 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 Bacillus caldolyticus and Bacillus stearothermophilus . There were only trace amounts of the protein in Thermotoga cells grown at 80°C. Therefore, to perform physicochemical experiments, the gene was cloned in Escherichia coli . A DNA probe was produced by PCR from genomic Tm DNA with degenerated primers developed from the known N‐terminus of Tm Csp and the known C‐terminus of CspB from Bacillus subtilis . Southern blot analysis of genomic Tm 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 csp 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 B. caldolyticus and high similarity to all other known Csps. Computer‐based homology modeling allowed the conclusion that Tm Csp represents a β‐barrel similar to CspB from B. subtilis and CspA from E. coli . As indicated by spectroscopic analysis, analytical gel permeation chromatography, and mass spectrometry, over‐expression of the recombinant protein yielded authentic Tm 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 Tm and represents the maximal T m ‐value reported for Csps so far.