Overexpression, oxidative refolding, and zinc binding of recombinant forms of the murine S100 protein MRP14 (S100A9)

Recombinant murine MRP14 (mMRP14) was produced in Escherichia coli using the pGEX expression system. The mass of fusion protein, by electrospray ionization-mass spectrometry (ESI/MS), was 39,213 Da which compares well with the theoretical mass (39,210.4 Da). Thrombin digestion of fusion protein was expected at a cloned thrombin consensus sequence(...LVP (R) under bar GS...) located between glutathione S-transferase and mMRP14, Analysis of products of digestion by C4 reverse-phase HPLC and SDS-PAGE/ Western blotting revealed two immunoreactive cleavage products with molecular weights around 13,000, Masses of the two proteins determined by ESI/MS were 13,062 and 11,919 Da. The larger product corresponded to the expected mass of recombinant mMRP14 (13,061.9 Da). Analysis of the protein sequence of recombinant mMRP14 revealed a thrombin-like consensus sequence (...NNP (R) under bar GH...) located close to the C-terminus. The smaller protein corresponded to a truncated form of rec mMRP14 (rec MRP14(1-102)) with a calculated mass of 11,918.6 Da. Optimization of the cleavage conditions resulted in >95% full-length rec mMRP14. Native mMRP14 contains one intramolecular disulfide bond between Cys(79) and Cys(90). The full-length recombinant protein was renatured and oxidized in ammonium acetate (pH similar to 7) for 96 h and formed >95% of the native intramolecular disulfide-bonded form. MRP14(1-102) bound substantially less Zn-65(2+) compared to native mMRP14 or rec mMRP14 after transfer to polyvinylidene difluoride and incubation with (ZnCl2)-Zn-65, implicating the His residues located within the C-terminal domain in Zn2+ binding. (C) 1999 Academic Press.