Abstract
Expression systems for highly toxic protein genes must be conditional and suppress leakage expression to almost zero because even faint leakage expression may kill host cells, inhibit host growth, and cause loss of plasmids containing the toxic protein genes. The most widely used conditional expression systems are controlled only at the transcriptional level, and complete suppression of leakage expression is challenging. Recent progress on translational control has enabled construction of dual transcriptional-translational control systems in which leakage expression is strongly suppressed. This review summarizes the principles, features, and practical examples of dual transcriptional-translational control systems in bacteria, and provides future perspectives on these systems.
Highlights
Heterologous gene expression is a fundamental technique for the production of recombinant proteins
HYZEL with translation of the toxic protein controlled by ZK incorporation tolerated an expression construct for colicin E3 enzymatic domain (ColE3e) containing a single amber stop codon insertion, which suggests that leakage expression is almost zero (Kato Y, unpublished data, Figure 2A), while the yield of recombinant protein was not affected by insertion of 1 or 2 amber stop codons [21]
A recombinant protein gene is transcribed by T7 RNA polymerase (T7RNP) and regulated by lactose operon in Escherichia coli (lacO/I) and PPDA-ORS. tT/tT achieved low leakage expression (0.1%) and high gain (850-fold), suggesting that tightness was increased by >240-fold by addition of PPDA-ORS regulation, whereas the volumetric yield decreased by about 60%
Summary
Heterologous gene expression is a fundamental technique for the production of recombinant proteins. A problem with this technique is that about 50% of artificially expressed proteins are toxic to the host bacteria, even in self-gene overexpression [5,6]. Loss of plasmids containing the toxic protein genes frequently occurs To overcome this problem, conditional expression systems, in which toxic proteins are produced only in the presence of inducers, are widely used [7]. The switchability of most “biological” conditional expression systems is not as perfectly tight as that of electronic switches This means a small, but problematic, number of proteins are produced, even in the absence of inducers (leakage expression) [8]. Complete suppression of leakage expression is essential for production of active highly toxic proteins The principles, features, and recent practical examples of dual transcriptional-translational control systems are described, together with a discussion of future perspectives on these systems
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