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Cloning, overexpression, purification, and physicochemical characterization of a cold shock protein homolog from the hyperthermophilic bacterium Thermotoga maritima
Author(s) -
Welker Christine,
Böhm Gerald,
Schurig Hartmut,
Jaenicke Rainer
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.2.394
Subject(s) - thermotoga maritima , cold shock domain , bacillus subtilis , biology , escherichia coli , gene , microbiology and biotechnology , genomic dna , biochemistry , recombinant dna , peptide sequence , sequence analysis , genetics , bacteria , rna
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.