PheBo Fusion Protein Cloning
A complete system for fusion cloning, cleavage, and purification

APPLICATIONS

• Protein purification from the cytoplasm or periplasm on affinity columns
• Inducible expression
• Inclusion bodies: formed only with extremely insoluble proteins; endoproteinase Pro-Pro-Y-Pro cleaves inclusion bodies during the affinity purification process, with no endoproteinase contamination.
• Optional protein export into the periplasm for correct folding

In the PheBo-System for fusion protein cloning and purification, the protein of interest is cloned next to the leader protein ß-lactamase. The expressed fusion protein remains in the cytoplasm (vectors pHKcyto1 and pHKcyto2) or is released into the periplasm with the help of a signal sequence (vectors pHKperi1 and pHKperi2). ß-lactamase allows efficient protein purification under mild conditions on pre-packed re-usable Phenylboronate (PheBo-) affinity columns. The leader protein is removed by the site-specific Endoproteinase Pro-Pro-Y-Pro (Igase), which also cleaves inclusion bodies. After a second passage through the same affinity column, the protein of interest can be obtained in pure form from the column flow-through, whereas ß-lactamase and the endoproteinase remain on the column.

References: Kolmar, K. et al., J.Mol.Biol. 228 (1992) 359-365. Frisch. C, et al., Biol.Chem.Hoppe-Seyler 375 (1994) 353-356.