High-level expression of soluble protein in Escherichia coli using a His6-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 expression vectors which contain a sequence encoding six consecutive histidine residues (His6-tag) at the 3' end of the MBP-encoding malE gene which is followed by either a thrombin-binding site (LVPRGS) or a factor Xa-binding site (IEGR). The benefits of this approach include; (a) high expression levels of soluble MBP fusion proteins (exceeding 2% of the total cellular protein), (b) high-quality purification of proteins under various conditions (high salt, low salt, denaturing, nondenaturing, etc.), and (c) two alternative protease cleavage sites to test for the most efficient cleavage of each fusion protein. We also constructed these MBP-His 6-tag expression vectors with alternative selection markers (Ampr, Kanr) and alternative promoters (tac, T7). Using these constructs, we expressed and purified several proteins of which we present two, penicillin-binding protein PBP2a and UDP-N-acetylmuramate:L-alanine ligase (MurC), and compare their expression level and purity with other expression systems. We also discuss the use of minimal media with supplements versus rich media and cell growth strategies to optimize the protein yield in general and for isotope labeling.
Pryor
K D
KD
Department of Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Leiting
B
B
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005822:Genetic Vectors; D006602:Hexosyltransferases; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D002267:Muramoylpentapeptide Carboxypeptidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D046915:Penicillin-Binding Proteins; D010453:Peptide Synthases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010458:Peptidyl Transferases; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D046915:Penicillin-Binding Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D006639:Histidine; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D010453:Peptide Synthases; C023229:UDP-N-acetylmuramoyl-alanine synthetase