Now that we understand what the numbers, letters and abbreviations mean in the Flexi® Vector naming convention, how does one choose from among the over 40 different vectors? First I will detail the uses for the 21 vectors in the 1–12 range (plus outlier pF25) and address the remaining vectors in a second entry.
- pF1 vectors are a basic vector for cloning a protein-coding sequence. The native (untagged) protein can be induced to express from the T7 promoter in E. coli systems or expressed in cell-free expression systems.
- pFN2 vectors have an N-terminal glutathione-S-transferase (GST) tag for purifying proteins. The cloned protein-coding region can be expressed from the T7 promoter in E. coli systems or in cell-free expression systems. If desired, the GST peptide can be cleaved from the fusion protein using TEV protease.
- pF3 vectors contain the 5´ and 3´ translation-enhancing sequences of Barley Yellow Dwarf Virus (BYDV), which can increase protein expression in wheat germ extracts. Therefore, protein-coding regions cloned into these vectors will express native (untagged) protein in wheat germ extract systems (e.g., Wheat Germ Extract Plus).
- pF4 vectors use the human cytomegalovirus (CMV) intermediate-early enhancer/promoter for constitutive native (untagged) protein expression in mammalian cells. These vectors also contain the T7 RNA polymerase promoter for cell-free protein expression, and can be used for both stable and transient gene expression; stable if cotransfected with a vector that has a selectable marker.
- pF5 vectors contain both the CMV promoter and the selectable marker neomycin (neo). Thus, native (untagged) proteins can be transiently or stably expressed in mammalian cells. With the T7 RNA polymerase promoter, proteins can also be used with E. coli or cell-free expression systems.
- pFN6 vectors are configured to append the Histidine-Glutamine (HQ) metal-binding peptide (MKHQHQHQAIA) to the amino terminus of protein-coding regions. Fusion proteins can be expressed in bacteria and cell-free expression systems and purified using the HQ tag and polyhistidine purification systems (e.g., HisLink™ Protein Purification Resin).
- pFC7 vectors are related to the pFN6 vectors and configured to append the HQ metal-binding peptide (VSHQHQHQ) to the carboxy terminus of a protein. As with the pFN6 vectors, fusion proteins can be expressed in bacteria and cell-free expression systems and purified with polyhistidine purification systems. Both the pFN6 and pFC7 vectors also have a T7 RNA polymerase promoter that can be used to induce expression of the fusion protein.
- pF9A CMV hRluc-neo Flexi® Vector can be used to express native protein in mammalian cells. Neomycin selects for stable transfectants, and the Renilla luciferase gene (hRluc) allows for reporter screening of transfected cells.
- pFN10A and pFN11A vectors are part of the Checkmate™/Flexi® Mammalian Two-Hybrid System and are used to clone the potential protein binding partners. The pFN10A vector contains a herpes simplex virus VP16 transcriptional activation domain while the pFN11A vector contains the yeast GAL4 DNA-binding domain, both upstream of the cloning site. Furthermore, the pFN11A vector also expresses Renilla reniformis luciferase under the control of the SV40 promoter for normalizing any differences in transfection efficiency.
- pF12 vectors are part of the Regulated Mammalian Expression System, and contain 12 copies of lambda operator sequences and a minimal CMV promoter upstream of the cloning site. This system regulates protein expression levels using varying concentrations of coumermycin while novobiocin acts as an antagonist to completely shut off protein expression.
- pF25 vectors contain a T7 polymerase promoter for expression of native proteins, and untranslated region (UTR) sequences at the 5´ and 3´ ends of the gene coding region to enhance translation efficiency in insect cell extract systems. These vectors are compatible with our TNT® T7 Insect Cell Extract Protein Expression System.
This vector listing covers about half of the Flexi® Vectors available. In my next post, I cover the remaining vectors, the HaloTag® Flexi® Vectors, and the potential uses in your research.
