Abstract:
: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.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Raftery MJ,Collinson L,Geczy CLdoi
10.1006/prep.1998.1015keywords:
subject
Has Abstractpub_date
1999-03-01 00:00:00pages
228-35issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(98)91015-4journal_volume
15pub_type
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