A complete system for expression, efficient protease cleavage and easy affinity purification

products | DNA vector systems | Fusion Protein Cloning System PheBo

DNA Vector Systems

Fusion Protein Cloning System PheBo

Features:

protein purification from the cytoplasm (pHKcyto)
preparative protein accumulation in the periplasm (pHKperi)
protein export into the periplasm for correct protein folding
endoproteinase Pro-Pro-Y-Pro cleaves also inclusion bodies!
contamination with the endoproteinase
Pro-Pro-Y-Pro avoided
very high yield
convenient protein purification via pre-packed affinity columns

Fusion Protein Expression and Purification:

Schematic overview of IPTG induced fusion protein expression, purification via PheBo column, endoproteinase cleavage of fusion protein and final purification of the protein of interest with a second application of the same PheBo column; note, that the endoproteinase Pro-Pro-Y-Pro (Igase) remains on the column as well.

Product Description:

In the PheBo-System for fusion protein cloning and purification, the protein of interest is cloned next to the leader protein beta-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).

Beta-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, 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 beta-lactamase and the endoproteinase remain on the column.

Vector Maps:

pHKperi1-Periplasmic protein production with signal sequence

pHKcyto1-Cytoplasmic protein production

ORDER INFORMATION, SHIPPING STORAGE:
order#	description	amount
PHKP01	pHKperi1 vector DNA, lyophilized	5 µg
PHKP02	pHKperi2 vector DNA, lyophilized	5 µg
PHKC01	pHKcyto1 vector DNA, lyophilized	5 µg
PHKC02	pHKcyto2 vector DNA, lyophilized	5 µg
PHKP12	pHKperi1 and pHKperi2	5 µg each
PHKC12	pHKcyto1 and pHKcyto2	5 µg each
PB0702	Phenylboronate column	2 ml
PB0705	Phenylboronate column	5 ml
shipped at RT; store at 4°C
EP0205	Endoproteinase Pro-Pro-Y-Pro (Igase)	50 µg
shipped on dry ice; store at -20°C

Downloads: Handbook

For further Vector System products please download the Vector Systems brochure (PDF, 3,9 MB)

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