Express and purify fusion proteins that are biotinylated in vivo. The DNA coding for the protein of interest is cloned into a PinPoint™ Vector downstream of a sequence encoding a peptide that becomes biotinylated in vivo. Biotinylated fusion proteins are produced in E. coli and are affinity-purified using the SoftLink™ Soft Release Avidin Resin. This proprietary resin allows elution of the fusion protein under nondenaturing conditions. The PinPoint™ Vectors feature the encoded endoproteinase Factor Xa (pronounced "ten a") proteolytic site that provides a way to separate the purification tag from the native protein, and the vectors carry a convenient multiple cloning region for ease in construction of fusion proteins.
The system contains vectors in all possible sense reading frames, an avidin-conjugated resin, Streptavidin-Alkaline Phosphatase, a purification column and biotin. The PinPoint™ Xa Control Vector contains the chloramphenicol acetyltransferase (CAT) gene provided as a means of monitoring protein expression, purification and processing conditions. The system generally yields 1–5mg of protein per liter of culture.
Additional features include the flexibility to use SoftLink™ Resin for release of the fusion protein under nondenaturing conditions and presence of the tac promoter, which allows tightly regulated expression.
- Purification of milligram amounts of expressed protein.
- Production of novel bioreagents.
- Cress, D. et al. (1993) Promega Notes 42, 2–7.