Monday, July 27, 2009 - 10:30 AM
S22
Molecular manipulation to improve structural stability of recombinant therapeutic protein
Lin Zhang, Murray Moo-Young, and C. Perry Chou. Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada
We demonstrated the enhancement of protein stability for the extracellular domain of human CD83 (hCD83ext) by manipulation of protein molecule and production host. NCBI Conserved Domain-search BLAST was used to identify the target amino acid residue for mutagenesis. Disulfide bonds were assigned in the protein product by peptide mass mapping based upon cyanylation of free cysteine residues with 1-cyano-4-dimethylamino-pyridinium (CDAP), followed by basic cleavage in aqueous ammonia (CN-induced cleavage). The cleavage mixture was subsequently analyzed by electrospray ionization quadruple time of flight mass spectrometry (ESI-QTOF MS). The role associated with disulfide bond formation, either intramolecular or intermolecular, for various cysteine residues were analyzed. This study highlights the overwhelming impact of disulfide pattern to the stability of the therapeutic protein. The strategy adopted herein will be useful for developing protein-based biopharmaceuticals that often encounter protein instability problems.
Keywords: CN-induced cleavage, disulfide bond, extracellular domain of human CD83 (hCD83ext), Escherichia coli, ESI-QTOF MS, peptide mass mapping, stability
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