title: Design of stability at extreme alkaline pH in streptococcal protein G.
abstract: Protein G (PrtG) is widely used as an affinity-based ligand for the purification of IgG. It would be desirable to improve the resistance of affinity chromatography ligands, such as PrtG, to commercial cleaning-in-place procedures using caustic alkali (0.5 M NaOH). It has been shown that Asn residues are the most susceptible at extreme alkaline pH: here, we show that replacement of all three Asn residues within the IgG-binding domain of PrtG only improves stability towards caustic alkali by about 8-fold. Study of the effects of increasing pH on PrtG by fluorescence and CD shows that the protein unfolds progressively between pH 11.5 and 13.0. Calculation of the variation in electrostatic free energy with pH indicated that deprotonation of Tyr, Lys and Arg side-chains at high pH would destabilize PrtG. Introduction of the triple mutation Y3F/T16I/T18I into PrtG stabilized it by an extra 6.8 kcal/mol and the unfolding of the protein occurred at a pH of about 13, or 1.5 pH units higher than wild type. The results show that strategies for the stabilization of proteins at extreme alkaline pH should consider thermodynamic stabilization that will retain the tertiary structure of the protein and modification of surface electrostatics, as well as mutation of alkali-susceptible residues.
authors.affiliation: Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Palmer
authors.forename: Katy
authors.initials: K
authors.lastname: Angus
authors.forename: Linda
authors.initials: L
authors.lastname: Taylor
authors.forename: Jim
authors.initials: J
authors.lastname: Warwicker
authors.forename: Jeremy P
authors.initials: JP
authors.lastname: Derrick
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine; D001426:Bacterial Proteins / Q000737:chemistry*; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008239:Lysine; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D055672:Static Electricity; D013816:Thermodynamics; D014443:Tyrosine
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D014443:Tyrosine; D001120:Arginine; D008239:Lysine