pMAL protein Fusion and Purification System

pMAL protein Fusion and Purification System

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The system uses the pMAL vectors which are designed so thatinsertion interrupts a lacZα gene allowing a blue-to-white screen forinserts on X-gal (5). pMAL-c2 series has an exact deletion of the malEsignal sequence, resulting in cytoplasmic expression of the fusionprotein. pMAL-p2 series contains the normal malE signal sequence,which directs the fusion protein through the cytoplasmic membrane.pMAL-p2 fusion proteins capable of being exported can be purifiedfrom the periplasm. Between the malE sequence and the polylinkerthere is a spacer sequence coding for 10 asparagine residues. Thisspacer insulates MBP from the protein of interest, increasing thechances that a particular fusion will bind tightly to the amylose resin.The vectors also include a sequence coding for the recognition site ofa specific protease. This allows the protein of interest to be cleavedfrom MBP after purification, without adding any vector-derivedresidues to the protein (6). For this purpose, the polylinker includes arestriction site superimposed on the sequence coding for the site ofthe specific protease. This is where the gene of interest is inserted. AnEcoR I site in the same reading frame as that of λgt11 and a numberof other useful sites are present directly downstream. The vectorsalso include the M13 origin of DNA replication which allows theproduction of single-stranded DNA for sequencing and mutagenesisby infecting with M13KO7 helper phage (NEB #N0315S

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