title: Positional effects of fusion partners on the yield and solubility of MBP fusion proteins.
abstract: Escherichia coli maltose-binding protein (MBP) is exceptionally effective at promoting the solubility of its fusion partners. However, there are conflicting reports in the literature claiming that (1) MBP is an effective solubility enhancer only when it is joined to the N-terminus of an aggregation-prone passenger protein, and (2) MBP is equally effective when fused to either end of the passenger. Here, we endeavor to resolve this controversy by comparing the solubility of a diverse set of MBP fusion proteins that, unlike those analyzed in previous studies, are identical in every way except for the order of the two domains. The results indicate that fusion proteins with an N-terminal MBP provide an excellent solubility advantage along with more robust expression when compared to analogous fusions in which MBP is the C-terminal fusion partner. We find that only intrinsically soluble passenger proteins (i.e., those not requiring a solubility enhancer) are produced as soluble fusions when they precede MBP. We also report that even subtle differences in inter-domain linker sequences can influence the solubility of fusion proteins.
authors.affiliation: Protein Engineering Section, Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Sreejith
authors.initials: S
authors.lastname: Raran-Kurussi
authors.affiliation: Protein Engineering Section, Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Karina
authors.initials: K
authors.lastname: Keefe
authors.affiliation: Protein Engineering Section, Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA. Electronic address: waughd@mail.nih.gov.
authors.forename: David S
authors.initials: DS
authors.lastname: Waugh
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine
keywords: Fusion protein; Gateway cloning; Inclusion bodies; MBP; Solubility enhancer