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 (ΔN-rmGIP) in E. coli Rosetta(DE3)-RIPL cells. Whereas ΔN-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 ΔN-rmGIP. The phosphatase activity of both proteins was Mg2+-dependent, with a pKMg of activation being ∼2.8 and non-cooperative binding. The Golli-myelin basic protein isoform rmBG21 (recombinant murine form) enhanced the phosphatase activity of ΔN-rmGIP below 6 μM, but significantly inhibited it at higher concentrations. Using glutaraldehyde cross-linking and gel shift assays, the rmBG21-ΔN-rmGIP interaction was shown to be equimolar and specific, but seemingly relatively weak, suggesting that a third interaction partner is required in vivo.