The potential of cold-shock promoters for the expression of recombinant proteins in microbes and mammalian cells.

BACKGROUND: Low-temperature expression of recombinant proteins may be advantageous to support their proper folding and preserve bioactivity. The generation of expression vectors regulated under cold conditions can improve the expression of some target proteins that are difficult to express in different expression systems. The cspA encodes the major cold-shock protein from Escherichia coli (CspA). The promoter of cspA has been widely used to develop cold shock-inducible expression platforms in E. coli. Moreover, it is often necessary to employ expression systems other than bacteria, particularly when recombinant proteins require complex post-translational modifications. Currently, there are no commercial platforms available for expressing target genes by cold shock in eukaryotic cells. Consequently, genetic elements that respond to cold shock offer the possibility of developing novel cold-inducible expression platforms, particularly suitable for yeasts, and mammalian cells. CONCLUSIONS: This review covers the importance of the cellular response to low temperatures and the prospective use of cold-sensitive promoters to direct the expression of recombinant proteins. This concept may contribute to renewing interest in applying white technologies to produce recombinant proteins that are difficult to express.

Evolutionary and structural constraints influencing apolipoprotein A-I amyloid behavior.

Apolipoprotein A-I (apoA-I) has a key function in the reverse cholesterol transport. However, aggregation of apoA-I single point mutants can lead to hereditary amyloid pathology. Although several studies have tackled the biophysical and structural consequences introduced by these mutations, there is little information addressing the relationship between the evolutionary and structural features that contribute to the amyloid behavior of apoA-I. We combined evolutionary studies, in silico mutagenesis and molecular dynamics (MD) simulations to provide a comprehensive analysis of the conservation and pathogenic role of the aggregation-prone regions (APRs) present in apoA-I. Sequence analysis demonstrated that among the four amyloidogenic regions described for human apoA-I, only two (APR1 and APR4) are evolutionary conserved across different species of Sarcopterygii. Moreover, stability analysis carried out with the FoldX engine showed that APR1 contributes to the marginal stability of apoA-I. Structural properties of full-length apoA-I models suggest that aggregation is avoided by placing APRs into highly packed and rigid portions of its native fold. Compared to silent variants extracted from the gnomAD database, the thermodynamic and pathogenic impact of amyloid mutations showed evidence of a higher destabilizing effect. MD simulations of the amyloid variant G26R evidenced the partial unfolding of the alpha-helix bundle with the concomitant exposure of APR1 to the solvent, suggesting an insight into the early steps involved in its aggregation. Our findings highlight APR1 as a relevant component for apoA-I structural integrity and emphasize a destabilizing effect of amyloid variants that leads to the exposure of this region.