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.

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