Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins.
It is difficult to imagine any strategy for high-throughput protein expression and purification that does not involve genetically engineered affinity tags. Because of its ability to enhance the solubility and promote the proper folding of its fusion partners, Escherichia coli maltose-binding protein (MBP) is a particularly useful affinity tag. However, not all MBP fusion proteins bind efficiently to amylose resin, and even when they do it is usually not possible to obtain a sample of adequate purity after a single affinity step. To address this problem, we endeavored to incorporate supplemental affinity tags within the framework of an MBP fusion protein. We show that both the nature of the supplemental tags and their location can influence the ability of MBP to promote the solubility of its fusion partners. The most promising configurations for high-throughput protein expression and purification appear to be a fusion protein with a biotin acceptor peptide (BAP) on the N-terminus of MBP and/or a hexahistidine tag (His-tag) on the C-terminus of the passenger protein.
Routzahn
Karen M
KM
Protein Engineering Section, Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, P.O. Box B, Frederick, Maryland 21702, USA.
Waugh
David S
DS
D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D019921:Biotinylation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D033902:Periplasmic Binding Proteins / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012995:Solubility; D013379:Substrate Specificity
D000345:Affinity Labels; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease