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Disulfide Bond Substitution by Directed Evolution in an Engineered Binding Protein
Author(s) -
Drevelle Antoine,
Urvoas Agathe,
HamidaRebaï Mériam Ben,
Van Vooren Gérard,
Nicaise Magali,
ValerioLepiniec Marie,
Desmadril Michel,
Robert Charles H.,
Minard Philippe
Publication year - 2009
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200800745
Subject(s) - protein engineering , neocarzinostatin , cysteine , chemistry , combinatorial chemistry , scaffold , phage display , covalent bond , protein folding , disulfide bond , hapten , biochemistry , biophysics , biology , dna , enzyme , peptide , genetics , antibody , organic chemistry , computer science , database
Breaking ties : The antitumour protein, neocarzinostatin (NCS), is one of the few drug‐carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed‐evolution approach starting from NCS‐3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide‐free NCS variants suitable for a variety of purposes, including intracellular applications.The chromoprotein neocarzinostatin (NCS) has been intensively studied for its antitumour properties. It has recently been redesigned as a potential drug‐carrying scaffold. A potential limit of this protein scaffold, especially for intracellular applications, is the presence of disulfide bonds. The objective of this work was to create a disulfide‐free NCS‐derived scaffold. A generic targeted approach was developed by using directed evolution methods. As a starting point we used a previously engineered NCS variant in which a hapten binding site had been created. A library was then generated in which cysteine Cys88 and Cys93 and neighbouring residues were randomly substituted. Variants that preserved the hapten binding function were selected by phage display and further screened by colony filtration methods. Several sequences with common features emerged from this process. The corresponding proteins were expressed, purified and their biophysical properties characterised. How these selected sequences rescued folding ability and stability of the disulfide‐free protein was carefully examined by using calorimetry and the results were interpreted with molecular simulation techniques.