The T2A Peptide

t2a peptide

t2a peptide

A group of 18-22 amino acid peptide sequences coding for foot-and-mouth disease virus (FMDV) 2A protein induce ribosomal skipping when inserted between two CDSs of proteins in eukaryotic cells. A position 19 proline in the 2A peptide sequence is not linked to the downstream protein via a peptide bond and translation of the 2A peptide sequence into the downstream protein stops. The separated polypeptides are then used as independent protein products due to the ribosomal skip mechanism. This is sometimes referred to as the self-cleaving peptide mechanism.

The 2A peptide allows the expression of heterologous and homologous genes at similar levels in a single transcript, making it an ideal molecular tool for linking multiple transgenes together to facilitate plant-based recombinant protein production. However, in vivo expression of heterologous proteins requires an efficient way to identify expressing colonies and verify the expression of both the target and control genes. We successfully established a method to rapidly and efficiently detect successful protein expression in the yeast Saccharomyces cerevisiae, using a simple polycistronic construct containing an FMDV 2A peptide sequence and an enhanced green fluorescent protein (eGFP).

The construct was introduced into pTrEno-PF, a vector that contains the FMDV protease site XbaI at both ends. The eGFP and the 17-amino acid EBOV epitope peptide were codon optimized and synthesized by Genscript (Piscataway, NJ). Both the 2A peptide sequence and eGFP were placed in frame with each other, and inserted into the pTrEno-PF vector via their XbaI sites. eGFP and EBOV mAbs were isolated from 14 kanamycin-resistant T0 transformants with 3 to 1 segregation ratio by protein A/G affinity assay on magnetic beads followed by western blot analysis.

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