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 (. LVPRGS. ) 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 (. NNPRGH. ) located close to the C-terminus. The smaller protein corresponded to a truncated form of rec mMRP14 (rec MRP141-102) 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 Cys79 and Cys90. The full-length recombinant protein was renatured and oxidized in ammonium acetate (pH approximately 7) for 96 h and formed >95% of the native intramolecular disulfide-bonded form. MRP141-102 bound substantially less 65Zn2+ compared to native mMRP14 or rec mMRP14 after transfer to polyvinylidene difluoride and incubation with 65ZnCl2, implicating the His residues located within the C-terminal domain in Zn2+ binding.
Cytokine Research Unit, School of Pathology, University of New South Wales, Kensington, New South Wales, 2052, Australia. mraftery@unsw.edu.au
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Raftery
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Collinson
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Geczy
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D040502:Calgranulin B; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli; D015870:Gene Expression; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D009418:S100 Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D000943:Antigens, Differentiation; D040502:Calgranulin B; D011993:Recombinant Fusion Proteins; D009418:S100 Proteins; D013917:Thrombin; D015032:Zinc