Part:BBa_K5143012

Polycistronic Expression in Saccharomyces cerevisiae Using P2A Peptide

Description

The self-cleaving 2A peptide, a short (18-22 amino acids) virally derived sequence, enables efficient polycistronic expression in eukaryotic systems by self-cleaving during translation. This peptide likely evolved to replace traditional IRES sequences due to its smaller size, thus benefiting viruses with more compact genomes. Among various 2A sequences from different viruses (e.g., foot-and-mouth disease virus, equine rhinitis A, Thosea asigna, porcine teschovirus-1), the porcine teschovirus 2A (P2A) demonstrated the highest cleavage efficiency in human, zebrafish, and adult mice cell lines. In research, the P2A peptide facilitates multicistronic gene expression without the need for multiple promoters, splicing signals, proteolytic cleavage sites (e.g., TEV), or IRESs. Compared to IRES, P2A is smaller and enhances translational efficiency, making it ideal for coexpressing large proteins in plasmids where insert size is a limitation. Additionally, P2A preserves the authenticity of the expressed protein sequence by avoiding the "scar" left by traditional cleavage methods. Both P2A peptides and IRES enable the simultaneous expression of multiple coding regions under a single promoter. The P2A peptide, cloned in frame with the protein coding sequences, allows cleavage after translation, whereas IRES, a noncoding region, facilitates ribosome binding independently of the 5'-cap. The specific choice between P2A and IRES depends on the project's requirements for upstream and downstream gene expression levels. Overall, P2A peptides offer a powerful tool for creating artificial polycistronic mRNA in eukaryotes, enabling multiple genes to be driven by a single promoter, thereby enhancing research and biotechnological applications.

Translation Process

• Step 1: The ribosome translates Protein A.
• Step 2: The ribosome encounters P2A, causing a "ribosome skipping" event.
• Step 3: Translation continues with Protein B.

This process results in the expression of two separate proteins from a single mRNA, without additional promoters or splicing signals. The use of P2A allows for efficient coexpression, ideal for plasmids where insert size is limiting. The diagram visually emphasizes how P2A mediates the separation of proteins during translation, facilitating the production of distinct proteins from a single transcript.

Construction

The P2A gene was synthesised and its nucleotide sequence optimised for DNA synthesis and expression in Saccharomyces cerevisiae .

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Liu, Z. et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193 (2017).

Mukherjee, M. & Wang, Z. Q. A well-characterized polycistronic-like gene expression system in yeast. Biotechnology and Bioengineering 120, 260–271 (2023).

Müntjes, K. et al. Establishing Polycistronic Expression in the Model Microorganism Ustilago maydis. Front Microbiol 11, 1384 (2020).