Abstract
Oligopeptide "2A" and "2A-like" sequences ("2As"; 18-25aa) are found in a range of RNA virus genomes controlling protein biogenesis through "recoding" of the host-cell translational apparatus. Insertion of multiple 2As within a single open reading frame (ORF) produces multiple proteins; hence, 2As have been used in a very wide range of biotechnological and biomedical applications. During translation, these 2A peptide sequences mediate a eukaryote-specific, self-"cleaving" event, termed "ribosome skipping" with very high efficiency. A particular advantage of using 2As is the ability to simultaneously translate a number of proteins at an equal level in all eukaryotic systems although, naturally, final steady-state levels depend upon other factors-notably protein stability. By contrast, the use of internal ribosome entry site elements for co-expression results in an unbalanced expression due to the relative inefficiency of internal initiation. For example, a 1:1 ratio is of particular importance for the biosynthesis of the heavy-chain and light-chain components of antibodies: highly valuable as therapeutic proteins. Furthermore, each component of these "artificial polyprotein" systems can be independently targeted to different sub-cellular sites. The potential of this system was vividly demonstrated by concatenating multiple gene sequences, linked via 2A sequences, into a single, long, ORF-a polycistronic construct. Here, ORFs comprising the biosynthetic pathways for violacein (five gene sequences) and β-carotene (four gene sequences) were concatenated into a single cistron such that all components were co-expressed in the yeast Pichia pastoris. In this review, we provide useful information on 2As to serve as a guide for future utilities of this co-expression technology in basic research, biotechnology, and clinical applications.
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