No abstract

No abstract

No abstract

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.

Members of bacterial Csp (cold-shock protein) family promote cellular adaptation to low temperature and participate in many other aspects of gene expression regulation through mechanisms that are not yet fully elucidated. Csp proteins interact with single-stranded nucleic acids and destabilize nucleic acid secondary structures. Some Csp proteins also act as transcription antiterminators in vivo and in vitro. Here, we selected a mutation in the cloned cspE gene that abolished CspEinduced transcription antitermination. In vitro, mutant CspE showed RNA binding activity similar to that of the wild-type CspE but was unable to destabilize nucleic acid secondary structures. Thus, nucleic acid melting ability of CspE and its transcription antitermination activity are correlated. In vivo, mutant cspE was functional with respect to up-regulation of expression of rpoS, but, unlike the wild-type cspE, it did not complement the cold-sensitive phenotype of the quadruple ⌬cspA⌬cspB⌬cspG⌬cspE deletion strain. Thus, the nucleic acid-melting activity of Csp is critical for its prototypical function of supporting low temperature survival of the cell.When an exponentially growing culture of Escherichia coli is shifted from 30 to 15°C, the cells exhibit a cold-shock response (1). This response is characterized by a transient arrest of cell growth during which a number of genes are induced, in contrast to a severe inhibition of general protein synthesis. Among the cold-shock proteins, the most prominent is CspA.1 Other cold-inducible proteins include transcription factor NusA (2), polynucleotide phosphorylase (3), initiation factor IF2 (4), RecA (5), histone-like protein H-NS (6), DNA gyrase (7), ribosomeassociated factors RbfA (8), and CsdA (9).In E. coli, there are nine Csp proteins, from CspA through CspI, of which CspA, CspB, CspG, and CspI are cold-shockinducible. CspC and CspE are constitutively produced at 37°C, whereas CspD is induced upon nutritional deprivation. The induction patterns of CspF and CspH are not known (for review see Refs. 10 and 11). CspA homologues are widely distributed in prokaryotes, and CspA is homologous to the cold-shock domain in human Y-box protein YB-1 and the Y-box proteins from other eukaryotes (for review see Refs. 12 and 13).None of the CspA homologues appear to be singularly responsible for cold-shock adaptation, because deletions in any one of the csp genes do not result in cold sensitivity. The double and triple deletion mutations in E. coli csps (⌬cspA⌬cspB, ⌬cspA⌬cspG, ⌬cspB⌬cspG, ⌬cspA⌬cspI, or ⌬cspA⌬cspB⌬cspG) did not result in cold sensitivity (14). In a triple deletion strain of ⌬cspA⌬cspB⌬cspG, CspE accumulated at low temperatures, suggesting that members of the CspA family may functionally substitute for each other during cold acclimation of cells (14).In addition to their apparent, but poorly understood role during cold-shock response, many cellular processes appear to respond to changes in Csp protein concentrations even at higher temperature. In E. coli, the presence of camphor lea...