Expression and purification of the active variant of recombinant murine Golli-interacting protein (GIP)--characterization of its phosphatase activity and interaction with Golli-BG21.
We have successfully expressed an active variant of recombinant murine GIP (rmGIP) with the N-terminal domain deletion (DeltaN-rmGIP) in E. coli Rosetta(DE3)-RIPL cells. Whereas DeltaN-rmGIP could be purified under native conditions, the purification of full-length rmGIP required denaturing conditions; and the yields were 31.4 mg and 7.4 mg per L of culture, respectively. Purity was at least 97% as assessed by HPLC. Both proteins exhibited a well-defined secondary structure composition as determined by circular dichroism spectroscopy, with a slightly higher ratio of helical and strand components in DeltaN-rmGIP. The phosphatase activity of both proteins was Mg(2+)-dependent, with a pK(Mg) of activation being approximately 2.8 and non-cooperative binding. The Golli-myelin basic protein isoform rmBG21 (recombinant murine form) enhanced the phosphatase activity of DeltaN-rmGIP below 6 microM, but significantly inhibited it at higher concentrations. Using glutaraldehyde cross-linking and gel shift assays, the rmBG21-DeltaN-rmGIP interaction was shown to be equimolar and specific, but seemingly relatively weak, suggesting that a third interaction partner is required in vivo.
Bamm
Vladimir V
VV
Departments of Molecular and Cellular Biology, Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada.
Harauz
George
G
D000818:Animals; D002384:Catalysis; D002942:Circular Dichroism; D051379:Mice; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
C499047:Mbp protein, mouse; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C483466:golli-interacting protein, mouse; D010749:Phosphoprotein Phosphatases