Search

The purpose of this research is to study the thermal unfolding of high concentration bovine Immunoglobulin G (IgG) under 26 different experimental conditions by Fourier Transform Infrared spectroscopy with improved purge conditions and software calculations. When bovine IgG (25-200 mg/mL) was thermally denatured between pH 4.0 and 8.0, it was observed that at 25 mg/mL concentration, the protein exhibited maximum thermal stability at pH 6.0 and 7.0 as evident from the apparent T(m) values. Increasing the concentration from 25 to 100 mg/mL at those pH values increased the thermal resistance of the protein by 2-3 °C. But, at 200 mg/mL, IgG showed a small decrease in its transition temperature. Presence of 100 mM Trehalose enhanced the T(m) values at all conditions and possibly prevented the complete loss of IgG as insoluble aggregates at higher temperatures. Second derivative plots were constructed to explain the conformational changes of IgG during thermal unfolding.

Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development.

Staphylococcus aureus (S. aureus) is a human pathogen associated with skin and soft tissue infections (SSTI) and life threatening sepsis and pneumonia. Efforts to develop effective vaccines against S. aureus have been largely unsuccessful, in part due to the variety of virulence factors produced by this organism. S. aureus alpha-hemolysin (Hla) is a pore-forming toxin expressed by most S. aureus strains and reported to play a key role in the pathogenesis of SSTI and pneumonia. Here we report a novel recombinant subunit vaccine candidate for Hla, rationally designed based on the heptameric crystal structure. This vaccine candidate, denoted AT-62aa, was tested in pneumonia and bacteremia infection models using S. aureus strain Newman and the pandemic strain USA300 (LAC). Significant protection from lethal bacteremia/sepsis and pneumonia was observed upon vaccination with AT-62aa along with a Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE) that is currently in clinical trials. Passive transfer of rabbit immunoglobulin against AT-62aa (AT62-IgG) protected mice against intraperitoneal and intranasal challenge with USA300 and produced significant reduction in bacterial burden in blood, spleen, kidney, and lungs. Our Hla-based vaccine is the first to be reported to reduce bacterial dissemination and to provide protection in a sepsis model of S. aureus infection. AT62-IgG and sera from vaccinated mice effectively neutralized the toxin in vitro and AT62-IgG inhibited the formation of Hla heptamers, suggesting antibody-mediated neutralization as the primary mechanism of action. This remarkable efficacy makes this Hla-based vaccine a prime candidate for inclusion in future multivalent S. aureus vaccine. Furthermore, identification of protective epitopes within AT-62aa could lead to novel immunotherapy for S. aureus infection.

The thermal melting of bovine serum albumin (BSA) in the presence of excipients like ethanol and sucrose was studied by circular dichroism spectroscopy at physiological pH in phosphate buffered saline. Calculated apparentTmvalues were used to assess the thermal stability using two state fitted experimental curves. 0.5 M sucrose stabilized the BSA indicated by the increase inTmof ∼8°C when compared to theTmof the same solution measured in the absence of sucrose. Conversely, in the presence of varying concentrations of ethanol (2–20%), the protein was destabilized by a decrease of ∽3–10°C ofTm. In the binary mixture of sucrose and ethanol, theTmvalues showed that ethanol dominantly destabilized BSA in the presence of sucrose, possibly by weakening the hydrophobic interactions in the protein.

Helicobacter pylori cysteine-rich proteins (Hcps) are disulfide-containing repeat proteins. The repeating unit is a 36-residue, disulfide-bridged, helix-loop-helix motif. We use the protein HcpB, which has four repeats and four disulfide bridges arrayed in tandem, as a model to determine the thermodynamic stability of a disulfide-rich repeat protein and to study the formation and the contribution to stability of the disulfide bonds. When the disulfide bonds are intact, the chemical unfolding of HcpB at pH 5 is cooperative and can be described by a two-state reaction. Thermal unfolding is reversible between pH 2 and 5 and irreversible at higher pH 5. Differential scanning calorimetry shows noncooperative structural changes preceding the main thermal unfolding transition. Unfolding of the oxidized protein is not an all-or-none two-state process, and the disulfide bonds prevent complete unfolding of the polypeptide chain. The reduced protein is significantly less stable and does not unfold in a cooperative way. During oxidative refolding of the fully reduced protein, all the possible disulfide intermediates with a correct disulfide bond are formed. Formation of "wrong" (non-native) disulfide bonds could not be demonstrated, indicating that the reduced protein already has some partial repeating structure. There is a major folding intermediate with disulfides in the second, third, and fourth repeat and reduced cysteines in the first repeat. Disulfide formation in the first repeat limits the overall rate of oxidative refolding and contributes about half of the thermodynamic stability to native HcpB, estimated as 27 kJ mol(-1) at 25 degrees C and pH 7. The high contribution to stability of the first repeat may be explained by the repeat acting as a cap to protect the hydrophobic interior of the molecule.

Ankyrin repeats (AR) are 33-residue motifs containing a beta-turn, followed by two alpha-helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5 degrees C both follow monoexponential kinetics with k(folding) = 28 sec(-1) and k(unfolding) = 0.9 sec(-1). Kinetic and equilibrium unfolding parameters at 5 degrees C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.