PinPoint™ Xa Protein Purification System

The PinPoint™ Xa Protein Purification System is designed for the production and purification of 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...

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The PinPoint™ Xa Protein Purification System is designed for the production and purification of 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 and is provided as a means of monitoring protein expression, purification and processing conditions. The system generally yields 1–5mg of protein per liter of culture.

Features - Benefits

• In vivo Biotinylation Tag: Allows purification of fusion proteins; many proteins produced have been soluble.
• Easy to Use: Purification of biotinylated proteins with the SoftLink™ Resin can be performed by column or batch purification.
• Easy Detection: Streptavidin Alkaline Phosphatase can be used to detect the biotinylated fusion protein in a pseudo-Western format to monitor purification.
• Flexible: PinPoint™ Vectors are supplied for all reading frames.
• Gentle Release Conditions: SoftLink™ Resin allows release of the fusion protein under nondenaturing conditions.
• tac Promoter: Allows tightly regulated expression.

Applications

• Purification of milligram amounts of expressed protein.
• Production of novel bioreagents.

For more information, see the Protocols & Applications Guide.

References

1. Cress, D. et al. (1993) Promega Notes 42, 2–7.

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