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Arginine substitution by alanine at the P1 position increases the selectivity of CmPI-II, a non-classical Kazal inhibitor
- Source :
- Biochemistry and Biophysics Reports, Vol 26, Iss , Pp 101008- (2021)
- Publication Year :
- 2021
- Publisher :
- Elsevier, 2021.
-
Abstract
- CmPI-II is a Kazal-type tight-binding inhibitor isolated from the Caribbean snail Cenchritis muricatus. This inhibitor has an unusual specificity in the Kazal family, as it can inhibit subtilisin A (SUBTA), elastases and trypsin. An alanine in CmPI-II P1 site could avoid trypsin inhibition while improving/maintaining SUBTA and elastases inhibition. Thus, an alanine mutant of this position (rCmPI-II R12A) was obtained by site-directed mutagenesis. The gene cmpiR12A was expressed in P. pastoris KM71H yeast. The recombinant protein (rCmPI-II R12A) was purified by the combination of two ionic exchange chromatography (1:cationic, 2 anionic) followed by and size exclusion chromatography. The N-terminal sequence obtained as well as the experimental molecular weight allowed verifying the identity of the recombinant protein, while the correct folding was confirmed by CD experiments. rCmPI-II R12A shows a slightly increase in potency against SUBTA and elastases. The alanine substitution at P1 site on CmPI-II abolishes the trypsin inhibition, confirming the relevance of an arginine residue at P1 site in CmPI-II for trypsin inhibition and leading to a molecule with more potentialities in biomedicine.
Details
- Language :
- English
- ISSN :
- 24055808
- Volume :
- 26
- Issue :
- 101008-
- Database :
- Directory of Open Access Journals
- Journal :
- Biochemistry and Biophysics Reports
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.b61a017b5334f2eab0b6f0f932ee0a6
- Document Type :
- article
- Full Text :
- https://doi.org/10.1016/j.bbrep.2021.101008