pHEK293 Ultra Expression Vectors overview

• Ultra-high expression of a protein of interest in HEK 293 cells
• Flexibility of a single- or double-vector system
• Protein expression up to 10 times higher than with CMV-derived promoters
• Ideal for production of biomedically relevant proteins, including monoclonal antibodies

E. coli is commonly used to express recombinant proteins in life science research. However, mammalian cell-based expression systems have several advantages over E. coli, especially for eukaryotic protein expression. Expression in mammalian cells allows for proper protein folding and post-translational modification—features that are key for proper protein function in vitro. pHEK293 Ultra Expression Vectors enable ultra-high, transient overexpression of a protein of interest in human HEK 293 cells. Please visit our tech note for data specific to the use of these vectors for producing monoclonal antibodies.

Principles of high expression with the TAR-Tat system

High levels of recombinant protein expression with pHEK293 Ultra Expression Vectors is accomplished by a transcriptional activation mechanism based on the HIV-1 virus TAR-Tat system. The TAR (transactivation response) element is an RNA sequence derived from HIV-1 that forms a stem loop structure at the 5' end of the viral RNA. Tat is an RNA-binding protein that binds to TAR and promotes transcription.

pHEK293 Ultra Expression Vector I and Vector II systems incorporate the TAR-Tat elements in order to provide ultra-high expression of your protein of interest. Both Vector I and Vector II systems incorporate the TAR sequence in the 5' untranslated region of the target gene, where it will not affect the amino acid sequence of the expressed target protein. For the Vector I system, a Tat cassette is included on the same vector; the Vector II system includes the Tat cassette on a separate Enhancer Vector, enabling greater control of the protein of interest's expression level. With both systems, expression of the Tat protein greatly increases the expression of the protein of interest.

• pHEK293 Ultra Expression Vector I (Cat. # 3390)—Obtain high expression with the convenience of a single-plasmid system
• pHEK293 Ultra Expression Vector II (Cat. # 3392)—Optimize expression levels by varying the ratio of Vector II and the included Enhancer Vector
  
  

Visualization of high expression using fluorescent proteins

In order to assess protein expression capabilities, both pHEK293 Ultra Expression Vector systems were used to introduce a fluorescent protein (AcGFP1) into adherent and suspension HEK 293 cells. For comparison, the same assay was conducted with a vector expressing AcGFP1 from a CMV promoter (pBApo-CMV DNA) and a high-expression vector from Company A. Constructs were made by inserting AcGFP1 into the appropriate vectors (details listed in the table at the end of this page).

Higher fluorescence intensity was observed for the pHEK293 systems (conditions 2, 3, and 4) than for either of the other two vectors (conditions 1 and 5). Additionally, a color change in suspension cells was readily apparent to the naked eye for cells transfected with the pHEK293 vectors.

Flow cytometry was used to quantify the expression levels of AcGFP1 in adherent and suspension cells. Cells transfected with pHEK293 Ultra Expression Vector I (conditions 2 and 7) and pHEK293 Ultra Expression Vector II (conditions 3, 4, 8, and 9) displayed fluorescence intensities 5–11 times higher than that obtained with a typical CMV promoter (conditions 1 and 6), and 2–7 times higher than Company A's high-expression vector (conditions 5 and 10). Additionally, cells transfected with the two-vector system displayed an expected increase in AcGFP1 expression when the amount of Enhancer Vector was raised from 0.008 µg (condition 3) to 0.2 µg (condition 4) for adherent cells, and from 0.24 µg (condition 8) to 6 µg (condition 9) for suspension cells.