Cloning & Expression

Bicep Expression Vectors

 

An optimized EMCV IRES offers the highest protein levels and stable expression.


BICEP (BICistronic Expression Plasmid) vectors contain an internal ribosomal entry site (IRES) element from the encephalomyocarditis virus (EMCV) for translation of two open reading frames (ORFs) from one bicistronic message (Figure 1). BICEP vectors are designed to drive transcription of the bicistronic message under control of the strong human cytomegalovirus (CMV) promoter regulatory region. IRESs are relatively short DNA sequences that can initiate RNA translation in a 5' cap-independent fashion. Placement of the IRES and a second gene of interest (ORF 2) downstream of the first target gene (ORF 1) allows co-expression of ORF 1 in a cap-dependent manner and ORF 2 in a cap-independent fashion, thus facilitating translation of two proteins from one mRNA transcript. BICEP vectors permit co-expression of two genes of interest or co-expression of a target gene with a selection marker such as neor. Currently, pBICEP-CMV vectors for transient and stable mammalian expression are offered with various combinations of the FLAG®, 3xFLAG®, and c-myc epitope tags.

Sigma's EMCV IRES outperforms the competition by more than 30-fold as shown in Figure 2. pBICEP-CMV-1 and pBICEP-CMV-2 contain neomycin phosphotransferase (neor), which confers antibiotic resistance to G 418 sulfate. The neor is translationally regulated by the IRES. Mammalian cells that grow in the presence of G 418 are found to express high levels of recombinant protein due to both the target gene and selection marker being translated from one bicistronic message (Figure 3). BICEP vectors for stable expression also lack a mammalian origin of replication, and as a result, form stable cell lines through genomic integration of plasmid DNA. BICEP vectors outperform the competition due to strategic placement of the EMCV IRES to optimize intercistronic length and expression levels of recombinant protein.1


Features & Benefits

  • Highest protein expression levels (Figure 2)
  • Stable cell line production without the concern of plasmid loss
  • Maintains high expression levels after numerous passages (Figure 3)
  • Choices for stable or transient expression
  • New vectors with two MCS regions for cloning your own stable selection marker or multiple genes of interest


Reference

  1. Attal, J., Theron, M.C., Puissant, C., Houdebine, L.M., Medline® Effect of intercistronic length on internal ribosome entry site (IRES) efficiency in bicistronic mRNA. Gene Expression, Volume 8, Issue 5-6, 1999, Pages 299-309.