Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems.

Diverse Gram-negative bacteria use type III secretion systems (T3SS) to translocate effector proteins into the cytoplasm of eukaryotic cells. The type III secretion apparatus (T3SA) consists of a basal body spanning both bacterial membranes and an external needle. A sensor protein lies at the needle tip to detect environmental signals that trigger type III secretion. The Shigella flexneri T3SA needle tip protein, invasion plasmid antigen D (IpaD), possesses two independently folding domains in vitro. In this study, the solution behavior and thermal unfolding properties of IpaD's functional homologs SipD (Salmonella spp.), BipD (Burkholderia pseudomallei), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) were examined to identify common features within this protein family. CD and FTIR data indicate that all members within this group are alpha-helical with properties consistent with an intramolecular coiled-coil. SipD showed the most complex unfolding profile consisting of two thermal transitions, suggesting the presence of two independently folding domains. No evidence of multiple folding domains was seen, however, for BipD, LcrV, or PcrV. Thermal studies, including DSC, revealed significant destabilization of LcrV, PcrV, and BipD after N-terminal deletions. This contrasted with SipD and IpaD, which behaved like two-domain proteins. The results suggest that needle tip proteins share significant core structural similarity and thermal stability that may be the basis for their common function. Moreover, IpaD and SipD possess properties that distinguish them from the other tip proteins.

IpaD localizes to the tip of the type III secretion system needle of Shigella flexneri.

Shigella flexneri, the causative agent of shigellosis, is a gram-negative bacterial pathogen that initiates infection by invading cells within the colonic epithelium. Contact with host cell surfaces induces a rapid burst of protein secretion via the Shigella type III secretion system (TTSS). The first proteins secreted are IpaD, IpaB, and IpaC, with IpaB and IpaC being inserted into the host cell membrane to form a pore for translocating late effectors into the target cell cytoplasm. The resulting pathogen-host cross talk results in localized actin polymerization, membrane ruffling, and, ultimately, pathogen entry. IpaD is essential for host cell invasion, but its role in this process is just now coming to light. IpaD is a multifunctional protein that controls the secretion and presentation of IpaB and IpaC at the pathogen-host interface. We show here that antibodies recognizing the surface-exposed N terminus of IpaD neutralize Shigella's ability to promote pore formation in erythrocyte membranes. We further show that MxiH and IpaD colocalize on the bacterial surface. When TTSS needles were sheared from the Shigella surface, IpaD was found at only the needle tips. Consistent with this, IpaD localized to the exposed tips of needles that were still attached to the bacterium. Molecular analyses then showed that the IpaD C terminus is required for this surface localization and function. Furthermore, mutations that prevent IpaD surface localization also eliminate all IpaD-related functions. Thus, this study demonstrates that IpaD localizes to the TTSA needle tip, where it functions to control the secretion and proper insertion of translocators into host cell membranes.

Spectroscopic and calorimetric analyses of invasion plasmid antigen D (IpaD) from Shigella flexneri reveal the presence of two structural domains.

Shigella flexneri is a facultative intracellular pathogen that causes severe gastroenteritis in humans. Invasion plasmid antigen D (IpaD) is an essential participant in Shigella invasion of intestinal cells, but no detailed structural information is available to help understand the proposed role of IpaD in invasion or its interaction with other invasion proteins. Therefore, the secondary and tertiary structure and thermal stability of IpaD as well as selected IpaD deletion mutants were investigated using Fourier transform infrared (FTIR), circular dichroism (CD), and both intrinsic and extrinsic fluorescence spectroscopies. The energetics of thermal unfolding were also evaluated by differential scanning calorimetry (DSC). Secondary-structure analysis by CD and FTIR suggests that that IpaD is primarily alpha-helical with characteristics of a intramolecular coiled coil. Thermal studies revealed that the unfolding of IpaD is a complex process consisting of two transitions centered near 59 and 80 degrees C. A comparison of the data obtained with the intact protein and selected deletion mutants indicated that the lower temperature transition is a reversible event attributable to the unfolding of a small domain located at the N terminus of IpaD. In contrast, the thermal unfolding of the proposed major and highly stable C-terminal domain was irreversible and led to protein aggregation. When the results are taken together, they strongly support the idea that IpaD has two independent folding domains.

Analytical and biological characterization of supercoiled plasmids purified by various chromatographic techniques.

Supercoiled plasmids are an important component of gene-based delivery vehicles. A number of production methods for clinical applications have been developed, each resulting in very high-quality product with low levels of residual contaminants. There is, however, no consensus on the optimal methods to characterize plasmid quality, and further, to determine if these methods are predictive of either product stability or biological activity. We have produced two plasmids using four production purification methodologies based on PolyFlo and hydrophobic interaction chromatography (HIC), either alone or in tandem processes. In each case, the product was analyzed using standard molecular biological methods. We also performed a number of biophysical analyses such as dynamic light scattering (DLS), circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). Minimal differences were detected among the preparations based on the more standard molecular biological methods. Some small differences were detected, however, using biophysical techniques, particularly FTIR and DSC, which may reflect small variations in plasmid tertiary structure and thermal stability. Stability after heat exposure at 60 degrees C, exposure to fetal bovine serum and long-term storage at 4 degrees C varied between plasmids. One plasmid showed no difference in stability depending on the production process, but the other showed significant differences. Evaluation in vivo in models for gene immunization and gene therapy showed significant differences in the response depending on the method of purification. Preparations using a tandem process of PolyFlo used in two separation modes provided higher biological activity compared to a tandem HIC/PolyFlo process or either resin used alone in a single column process. These data indicate that the process by which supercoiled plasmids are made can influence plasmid stability and biological activity and emphasize the need for more rigorous methods to evaluate supercoiled plasmids as gene-delivery vehicles.