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

Abstract

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.

Keywords: Active enzyme; Aggregation-prone recombinant protein; Cold-inducible promoter; Protein quality; Protein stability; Unstable gene product.

Fig. 1

Structure of prokaryotic and eukaryotic genes. A and B show the elements that comprise the canonical genes. A In prokaryotic genes, the regulatory gene elements for transcription are sited upstream, downstream, and on regulatory sequences such as RBS and intergenic UTRs. B In eukaryotic genes, the regulatory gene elements for transcription are sited upstream, downstream, and intronic regions. 5′-UTR: 5′-untranslated region, 3′-UTR: 3′-untranslated region, RBS: ribosome binding site

Fig. 2

Effect of sub-physiological temperatures on CSP translation. A Protein translation at physiological temperature where the concentration of non-CSPs increases while the CSPs remain at low levels. B The cellular response under cold shock involves modulation of protein expression where the concentration of CSPs increases while the non-CSPs diminishes. Reduced expression of non-CSPs is linked to repression of non-csp mRNA translation, leading to slower growth and cell cycle arrest. CSP overexpression is linked to higher csp-mRNA translation due to a secondary structure change that facilitates ribosome docking. CSP overexpression allows cell growth to continue under cold stress. CSP: cold-shock protein

Fig. 3

Schematic representation of an expression module for a cold-shock vector. Cold-shock proteins (CSPs) are found in all organisms. The promoters of upregulated csp genes, ribosome binding site (RBS), and transcription terminator are the primary genetic elements regulating cold shock-directed expression. The two former elements are sited on the upstream csp 5′-end, whereas the third element is sited downstream on the csp 3′-end. In addition to these genetic elements, the expression module must also contain the translation start codon (ATG), the multiple cloning sites (MCS), and the translation stop codon (Stop). Optionally, it can have some tags, such as the 6xHis tag, to facilitate the purification of recombinant proteins. csp 5′-UTR: 5′-untranslated region of csp, csp 3′-UTR: 3′-untranslated region of csp. Adapted from Bjerga and Williamson 2015