Using 2A “self-cleaving” peptide in bicistronic mammalian expression

Multiple promoters or internal ribosomal entry sites (IRES) have been used for the production of multiple proteins from the same vector. Potential drawbacks with multiple promoters on viral vectors include unstable genome and interference between promoters. IRES is a relatively large sequence that can cause problems in virus packaging, especially for viruses with very limited genome size such as AAV. In addition, it is required that the start of the second ORF is fairly close to the IRES, adding difficulties to cloning.

2A or 2A-like peptide (collectively called 2A peptide here) is used by several families of viruses, the best known foot-and-mouth disease virus of the Picornaviridae family, for producing multiple polypeptides. Although called a “self-cleaving” peptide or protease site, the mechanism by the 2A sequence for generating two proteins from one transcript is by ribosome skipping–a normal peptide bond is impaired at 2A, resulting in two discontinuous protein fragments from one translation even.

The 2A-based bicistronic expression has been used for several years, but recently gained much more popularity due to its successful use in iPSC generation that required 2 to 4 factors working in concert. Even expression of all factors can be achieved when 2A peptides are used for multiple protein production, due to near 100% efficiency of the 2A “cleavage” at each site, and no interference between multiple 2A sites. Early work used a 36 amino acid sequence as 2A peptide, which was later reduced to about half that size from mutation and screening. Commercial vectors utilizing 2A for co-expression of cDNA and fluorescent protein and/or drug resistance genes have not been available until now. Allele Biotech has introduced a number of such plasmids, establishing another First-to-the-market as it has done many times previously in its 10 year history.

    New product of the week 03-29-10 to 04-04-10:

Alleleustrious pmTFP1-2A Bicistronic mammalian expression vector, ABP-FP-T2A10, $399,

    Promotion of the week 03-29-10 to 04-04-10:

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Introducing Baculo Virus Expression System (BVES) with a Strong IRES

Internal ribosome entry site (IRES) can be used to initiate translation of a second open reading frame (ORF) of an mRNA, providing the benefits of: 1) avoiding promoter competition in a dual promoter situation; 2) having controlled ratio of expression of two proteins; 3) placing a dominant selection pressure on the entire bicistronic mRNA and hence the maintenance of the transgene when a selection marker is placed as the second ORF.

IRES elements are located mainly in RNA viruses except certain mammalian and insect mRNA molecules. Only one DNA virus has so far been found to contain an IRES, the while spot syndrome virus (WSSV) of marine shrimp. This IRES, compared to a very few other choices known to function in insect cells such as the IRES from Rhopalosiphum padi virus (RhPV), has strong translation initiation activity (~98-99% in reference to cap-dependent initiation), insect cell specificity, and encompasses only 180 base pairs.

Allele Biotech, with its acquisition of Orbigen, is a major provider of BVES products and services with more than 10 years of experience. Allele’s featured New Products of the Week* this week are WSSV IRES containing baculovirus vectors, the sIRES (for Strong IRES from Shrimp virus) series plasmids. Currently one version is pOrb-MCS-sIRES-VSVG for pseudotyping baculoviruses (within the Emerald Baculovirus for Mammalian Expression series), with pOrb-mWasabi-sIRES-VSVG as a fluorescent protein control; the other is pOrb-MCS-sIRES-MCS for cloning a custom second cDNA. New versions in the future will include IRES driven mWasabi and other commonly used selection markers.

With a current research project for the National Cancer Institute (NCI) within the National Institutes of Health (NIH) involving development of modified BVES and mammalian protein expression and purification systems, Allele Biotech expects this product line to continue its expansion at a fast pace.

* Allele Biotech announces at least one new product every Wednesday through news release at AlleleNews or Allele Blog and social networks.

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Wednesday, September 30th, 2009 Viruses and cells No Comments