Complete system for expression of N-terminal authentic proteins, cleavage and easy protein purification

products | DNA vector systems | Fusion Protein Cloning System pAX

DNA Vector Systems

Fusion Protein Cloning System pAX

Features

expression of N-terminal authentic proteins without additional amino acids (pAX5)
cloning in three different reading frames (pAX4a, b, c)
production of both single DNA strands for sequencing the gene (f1 origin in + and - orientation)
purification of fusion proteins via pre-packed APTG affinity columns
cleavage of fusion protein with endoproteinase Ile-Glu-Gly-Arg (Xa) or collagenase
mutagenesis

Schematic overview of fusion protein expression, purification via APTG column, endoproteinase Ile-Glu-Gly-Arg cleavage of fusion protein and final purification of the protein of interest with a second application of the same APTG column.

Product Description:

The pAX vector system combines the advantages of optimal gene expression with those of simple protein purification via APTG affinity columns.

The fusion protein contains a rigid collagen structure between the leader protein beta -galactosidase and the foreign protein of interest, allowing both proteins to fold into their active forms independently. The protein hinge region also provides free access to the cleavage site of the endoprotease Ile-Glu-Gly-Arg (Factor Xa).

After expression, the fusion protein is separated from the cell lysate via APTG affinity chromatography. Proteolytic cleavage of the eluted fusion protein is performed either by endoproteinase Ile-Glu-Gly-Arg or collagenase. The former results in the release of a 5´ authentic foreign protein, if the gene is cloned into the Nru I site of pAX5.

A second application of the same APTG column separates the leader sequence (consisting of beta -galactosidase and collagen) from the cloned protein. The purified protein of interest is collected in the flow-through.

The pAX system can be used to clone, sequence, mutagenize and express foreign genes in E.coli and purify the gene product. Eight cloning vectors, which differ in the multiple cloning site, the reading frame and the orientation of the f1 origin, are available, as well as sequencing primers, endoproteinase Ile-Glu-Gly-Arg (see chapter 6 of our online catalog).

Host Strains:

E.coli lacI^q strains like JM109 are recommended, or strains which do not express beta -galactosidase. We suggest the helper phage M13K07.

Vector Map:

ORDER INFORMATION, SHIPPING & STORAGE:<
order#	description	amount
PAX4AP	pAX4a+vector DNA, lyophilized	10 µg
PAX4AM	pAX4a-vector DNA, lyophilized	10 µg
PAX4BP	pAX4b+vector DNA, lyophilized	10 µg
PAX4BM	pAX4b-vector DNA, lyophilized	10 µg
PAX4CP	pAX4c+vector DNA, lyophilized	10 µg
PAX4CM	pAX4c-vector DNA, lyophilized	10 µg
PAX5P	pAX5+vector DNA, lyophilized	10 µg
PAX5M	pAX5-vector DNA, lyophilized	10 µg
AXPRI1	pAX 5 ´ sequencing primer, lyophilized	0.05 A ₂₆₀
AXPRI2	pAX 3 ´ sequencing primer, lyophilized	0.05 A ₂₆₀
EP0501*	Endoproteinase IIe-Glu-Gly-Arg (Xa)	100 µg
EP0504*	Endoproteinase IIe-Glu-Gly-Arg (Xa)	250 µg
EP0502*	Endoproteinase IIe-Glu-Gly-Arg (Xa)	1 mg
shipped at RT; store at 4°C; * not available in the US

See also chapter 4 of our online catalog.

Downloads: Handbook
The DNA sequences are available on request.

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