The vector pMAL-p5G is designed to produce maltose-binding protein (MBP) fusions, where the protein of interest can be cleaved from MBP with the specific protease Genenase™ I (NEB #P8075).
MBP fusions made with this vector include an N-terminal signal sequence, so the fusion protein is directed to the periplasm. The MBP has been engineered for tighter binding to amylose resin.
A gene or open reading frame is inserted into a restriction site of the vector polylinker, in the same translational reading frame as the malE gene (encoding MBP). The fusion protein produced from the vector can be purified by amylose affinity chromatography. The sequence coding for the five amino acids Pro-Ala-Ala-His-Tyr is present just upstream of the SnaBI site. This allows the protein of interest to be cleaved from MBP with the specific protease Genenase™ I.
pMAL-p5G cut with SnaBI produces a blunt end at the tyrosine codon. This allows blunt-end cloning of an insert where the first three nucleotides code for the first amino acid of the protein of interest, and Genenase™ I cleavage of the fusion produces a protein with no vector-derived amino acids.
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