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

Can be used to:

• Clone in three different reading frames (pAX4a,b,c)
• Produce ssDNA in + and - orientation
• Purify fusion proteins on pre-packed APTG affinity columns
• Cleave fusion proteins with Endoproteinase Ile-Glu-Gly-Arg or Collagenase
• mutagenize

The pAX vector system combines the advantages of optimal gene expression and simple protein purification via APTG affinity columns. The fusion protein contains a rigid collagen structure between the leader protein, ß-galactosidase, and the foreign protein of interest, allowing both proteins to fold into active forms independently. The protein hinge region also provides free access to the cleavage site of the endonuclease Ile-Glu-Gly-Arg (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 foreign protein with an authentic 5' end in cases where the gene is cloned into the NruI site of pAX5. A second application of the same APTG column separates the leader sequence (consisting of ß-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. Additionally, as sequencing primers, endoproteinase Ile-Glu-Gly-Arg, and pre-packed APTG-columns are available as optional reagents.

endoprotease cleavage site, Xa transcription termination, to