title: Improved solubility of replication factor C (RFC) Walker A mutants.
abstract: Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously documented. We found that mutant forms of RFC harboring a single point mutation in the Walker A motif were even less soluble than the wild-type complex. The addition of maltose at 0.75 M to the storage and assay buffers greatly increases protein solubility and prevents the complex from falling apart. Our analysis of the clamp loading reaction is dependent on fluorescence-based assays, which are environmentally sensitive. Using wt RFC as a control, we show that the addition of maltose to the reaction buffers does not affect fluorophore responses in the assays or the enzyme activity, indicating that maltose can be used as a buffer additive for further downstream analysis of these mutants.
authors.affiliation: University of Florida, Department of Biochemistry & Molecular Biology, 1600 SW Archer Road, P.O. Box 100245, Gainesville, FL 32610-0245, USA.
authors.forename: Melissa R
authors.initials: MR
authors.lastname: Marzahn
authors.forename: Linda B
authors.initials: LB
authors.lastname: Bloom
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs; D002850:Chromatography, Gel; D008320:Maltose / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D017354:Point Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051818:Replication Protein C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D008320:Maltose; D000255:Adenosine Triphosphate; D051818:Replication Protein C