Characterization and stabilization of recombinant human protein pentraxin (rhPTX-2).

The potential human therapeutic protein pentraxin (PTX-2) is a large, glycosylated plasma protein consisting of five monomers that self-associate noncovalently into a pentameric, ring-like structure. The structural integrity and conformational stability of recombinant human PTX-2 (rhPTX-2) as a function of temperature and pH is described in conjunction with the identification of stabilizing excipients that improve the protein's physical stability. The monomer consisted primarily of two glycosylated forms (25,171 and 25,462 Da) as determined by mass spectrometry. The protein was approximately 82%-97% pentamer in solution with smaller amounts of decamer and aggregate as measured by analytical ultracentrifugation and size-exclusion chromatography (SEC). Comprehensive biophysical characterization using an empirical phase diagram approach demonstrates that rhPTX-2 is conformationally stable over a wide pH range (6.0-8.5) and at temperatures below 65°C-75°C. Formation of soluble aggregates was identified as a major physical degradation pathway of rhPTX-2 in solution under accelerated storage conditions. A series of effective stabilizers was identified by SEC analysis including disaccharides, sugar alcohols, citrate, as well as neutral and charged amino acids. The preformulation characterization data and stability profile presented in this work enable future studies for rhPTX-2 formulation development.

Engineering human IL-18 with increased bioactivity and bioavailability.

Cytokines in plasmid form can act as potent adjuvants when co-administered with DNA vaccines, resulting in an enhanced immune response to the DNA-encoded antigen. This is true of interleukin-18 (IL-18), which has been shown to serve as an adjuvant in conjunction with certain DNA vaccines. To determine if the properties of IL-18 could be optimized for use as a DNA vaccine adjuvant, a model of IL-18/IL-18R binding was developed to identify variants of human IL-18 that were predicted to improve receptor interactions and potentially bioactivity. The linkage of mature IL-18 to a secretion signal sequence provided improved protein expression from mammalian cells and signal peptidase cleavage of this protein produced the authentic N-terminus. The IL-18 variant proteins secreted this way were bioactive, as demonstrated by their ability to induce interferon gamma (IFNgamma) expression by human peripheral blood mononuclear cells (PBMCs) and to bind to IL-18R, as demonstrated by BIAcore analysis. The IL-18 variants were inhibited by IL-18 binding protein (IL-18BP), the soluble inhibitor of IL-18, as measured by neutralization of the IFNgamma response in PBMCs. One variant, V11I/T63A, demonstrated increases both in bioactivity and mammalian cell expression as compared to native IL-18, indicating that this molecule may be particularly well suited for use as a DNA-encoded vaccine adjuvant.

Entropy-enthalpy compensation in ionic interactions probed in a zinc finger peptide.

Zinc(II) and cobalt(II) binding to a series of zinc finger peptides with different charged residue pairs across from one another in a beta-sheet were examined. Previous studies revealed a narrow range of interaction free energies (<0.5 kcal/mol) between these residues. Here, isothermal titration calorimetry studies were performed, revealing a range of over 3 kcal/mol in relative binding enthalpies. Double mutant cycle analysis revealed a range of interaction enthalpies ranging from -3.1 to -3.4 kcal/mol for the Arg-Asp pair to -0.8 kcal/mol for the Lys-Glu pair. The large range of interaction enthalpies coupled with the small range of interaction free energies reveals substantial entropy-enthalpy compensation. The magnitudes of the effects are consistent with the formation of a structurally rigid Arg-Asp contact ion pair but less direct and more mobile interactions involving the other combinations.

Kinetics and thermodynamics of copper(II) binding to apoazurin.

The binding of copper(II) to apoazurin has been probed by isothermal titration calorimetry in cholamine buffer at pH 7.0. The standard enthalpy change was determined to be -10.0 +/- 1.4 kcal/mol. Each calorimetric trace reveals an initial exothermic phase followed by an endothermic phase. The calorimetric data could be fit to a kinetic model involving a bimolecular combination of copper(II) and apoazurin in an exothermic process (k = 2 +/-1 x 103 M-1 s-1, DeltaH degrees = -19 +/- 3 kcal/mol) to form an intermediate that spontaneously converts to Cu(II)-azurin in an endothermic process (k = 0.024 +/- 0.01 s-1, DeltaH degrees = +9 +/- 3 kcal/mol). These data suggest that copper(II) first combines with apoazurin in an irreversible process to form an intermediate that converts to copper(II)-azurin in a process driven by the release of water. The overall standard free energy of copper(II) binding to apoazurin is estimated to be -18.8 kcal/mol.

Structure-based thermodynamic analysis of a coupled metal binding-protein folding reaction involving a zinc finger peptide.

The thermodynamics of metal binding by the prototypical Cys(2)His(2) zinc finger peptide CP-1 has been examined through the use of isothermal titration calorimetry. In cholamine buffer at pH 7.0, the binding of zinc(II) to CP-1 shows an enthalpy change of DeltaH degrees (obs) = -33.7 +/- 0.8 kcal/mol. Between one and two protons appear to be released accompanying the metal binding process. The heat of protonation of the cholamine buffer used is quite large (-11.5 kcal/mol), indicating that a portion of the observed metal binding enthalpy is due to buffer protonation. Structure-based thermodynamic analysis including the effect of water release from zinc(II) appears to account for the entropy associated with the coupled metal binding-protein folding process semiquantitatively. The strongest driving force for the reaction is the enthalpy associated with the four bonds from zinc(II) to cysteinate and histidine residues, compared with the bonds from zinc(II) to water. The binding of cobalt(II) to CP-1 is less enthalpically driven than the binding of zinc(II) by -7.6 kcal/mol. This value is approximately equal to, but slightly larger than, the expectation based on considerations of ligand field stabilization energy.