title: Over-expression in E. coli and purification of functional full-length murine small C-terminal domain phosphatase (SCP1, or Golli-interacting protein).
abstract: During myelination in the central nervous system, proteins arising from the gene in the oligodendrocyte lineage (golli) participate in diverse events in signal transduction and gene regulation. One of the interacting partners of the Golli-isoform BG21 was discovered by yeast-2-hybrid means and was denoted the Golli-interacting-protein (GIP). In subsequent in vitro studies of recombinant murine GIP, it was not possible to produce a full-length version of recombinant murine rmGIP in functional form under native conditions, primarily because of solubility issues, necessitating the study of a hexahistidine-tagged, truncated form ΔN-rmGIP. This protein is an acidic phosphatase belonging to the family of RNA-polymerase-2, small-subunit, C-terminal phosphatases (SCP1), and studies of the human ortholog hSCP1 have also been performed on truncated forms. Here, a new SUMO-expression and purification protocol has been developed for the preparation of a functional, full-length mSCP1/GIP (our nomenclature henceforth), with no additional purification tags. Both full-length mSCP1/GIP and the truncated murine form (now denoted ΔN-rmSCP1/GIP) had similar melting temperatures, indicating that the integrity of the catalytic core per se was minimally affected by the N-terminus. Characterization of mSCP1/GIP activity with the artificial substrate p-NPP (p-nitrophenylphosphate) yielded kinetic parameters comparable to those of ΔN-rmSCP1/GIP and the truncated human ortholog ΔN-hSCP1. Similarly, mSCP1/GIP dephosphorylated a more natural CTD-peptide substrate (but not protein kinase C-phosphorylated BG21) with comparable kinetics to ΔN-hSCP1. The successful production of an active, full-length mSCP1/GIP will enable future evaluation of the functional role of its N-terminus in protein-protein interactions (e.g., BG21) that regulate its phosphatase activity.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Sergio
authors.initials: S
authors.lastname: Jaramillo-Tatis
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Bamm
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Kenrick A
authors.initials: KA
authors.lastname: Vassall
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada. Electronic address: gharauz@uoguelph.ca.
authors.forename: George
authors.initials: G
authors.lastname: Harauz
mesh_terms: D000818:Animals; D002490:Central Nervous System / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005786:Gene Expression Regulation; D051379:Mice; D004676:Myelin Basic Protein / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009578:Nitrobenzenes / Q000378:metabolism; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: C440032:4-nitrophenylphosphocholine; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins; D009578:Nitrobenzenes; D009687:Nuclear Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C483466:golli-interacting protein, mouse; D010767:Phosphorylcholine
keywords: Golli (gene in the oligodendrocyte lineage); Golli-interacting protein; Intrinsically-disordered protein; Myelin basic protein; Myelination; Oligodendrocyte; Phosphorylation; Small C-terminal domain phosphatase