The vector pMAL-c5X is designed to produce maltose-binding protein (MBP) fusions, where the protein of interest can be cleaved from MBP with the specific protease Factor Xa (NEB #P8010).
MBP fusions made with this vector are expressed cytoplasmically. 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 maltose-binding protein). The fusion protein thus produced can be purified by amylose affinity chromatography. The sequence coding for the four amino acids Ile-Glu-Gly-Arg is present just upstream of the XmnI site. This allows the protein of interest to be cleaved from maltose-binding protein with the specific protease Factor Xa. Fragments inserted in the XmnI site (cleaves GAAGG↓ATTTC) will produce a fusion protein that, after Factor Xa cleavage, contains no vector-derived residues on the protein of interest.
