HIGH-LEVEL EXPRESSION OF SOLUBLE-PROTEIN IN ESCHERICHIA-COLI USING A HIS(6)-TAG AND MALTOSE-BINDING-PROTEIN DOUBLE-AFFINITY FUSION SYSTEM

Using the maltose-binding protein (MBP) fusion vector pMAL-c1 from C. V. Maina et al. (1988, Gene 74, 365-373), we have constructed expressi on vectors which contain a sequence encoding six consecutive histidine residues (His(6)-tag) at the 3' end of the MBP-encoding malE gene whi ch is followed by either a thrombin-binding site (LVPRGS) or a factor Xa-binding site (IEGR). The benefits of this approach include: (a) hig h expression levels of soluble MBP fusion proteins (exceeding 2% of th e total cellular protein), (b) high-quality purification of proteins u nder various conditions (high salt, low salt, denaturing, nondenaturin g, etc.), and (c) two alternative protease cleavage sites to test for the most efficient cleavage of each fusion protein. We also constructe d these MBP-His(6)-tag expression vectors with alternative selection m arkers (Amp(r), Kan(r)) and alternative promoters (tac, T7). Using the se constructs, we expressed and purified sever al proteins of which we present two, penicillin-binding protein PBP2a and UDP-N-acetylmuramat e:L-alanine ligase (MurC), and compare their expression level and puri ty with other expression systems. We also discuss the use of minimal m edia with supplements versus rich media and cell growth strategies to optimize the protein yield in general and for isotope labeling. (C) 19 97 Academic Press.