Non-monotonic variation with salt concentration of the second virial coefficient in protein solutions

The osmotic virial coefficient $B_2$ of globular protein solutions is calculated as a function of added salt concentration at fixed pH by computer simulations of the ``primitive model''. The salt and counter-ions as well as a discrete charge pattern on the protein surface are explicitly incorporated. For parameters roughly corresponding to lysozyme, we find that $B_2$ first decreases with added salt concentration up to a threshold concentration, then increases to a maximum, and then decreases again upon further raising the ionic strength. Our studies demonstrate that the existence of a discrete charge pattern on the protein surface profoundly influences the effective interactions and that non-linear Poisson Boltzmann and Derjaguin-Landau-Verwey-Overbeek (DLVO) theory fail for large ionic strength. The observed non-monotonicity of $B_2$ is compared to experiments. Implications for protein crystallization are discussed.Comment: 43 pages, including 17 figure

Towards Protein Crystallization as a Process Step in Downstream Processing of Therapeutic Antibodies: Screening and Optimization at Microbatch Scale

Crystallization conditions of an intact monoclonal IgG4 (immunoglobulin G, subclass 4) antibody were established in vapor diffusion mode by sparse matrix screening and subsequent optimization. The procedure was transferred to microbatch conditions and a phase diagram was built showing surprisingly low solubility of the antibody at equilibrium. With up-scaling to process scale in mind, purification efficiency of the crystallization step was investigated. Added model protein contaminants were excluded from the crystals to more than 95%. No measurable loss of Fc-binding activity was observed in the crystallized and redissolved antibody. Conditions could be adapted to crystallize the antibody directly from concentrated and diafiltrated cell culture supernatant, showing purification efficiency similar to that of Protein A chromatography. We conclude that crystallization has the potential to be included in downstream processing as a low-cost purification or formulation step

MPCD : A new interactive-on-line crystallization data bank for screening strategies

The Marseille Protein Crystallization Database (MPCD) is a new crystallization database, freely accessible via http://www.crmcn.univ-mrs.fr/ mpcd/, populated with information found in the BMCD and CYCLOP. This new online database includes details about macromolecules (such as name, pI, molecular weight, number of subunits), crystallization conditions, methods and additives used in an easy-to-compare table form. It allows users to choose their own crystallization parameters, to create tables for further analysis and also to enter new proteins and crystallization conditions in order to supplement this database. As an essential tool in structural biology, this crystallization database will be highly relevant to crystal growers, when connected to the Protein Data Bank, for the crystallogenesis of a variety of structurally distinct molecules and assemblies, and to macromolecular and biomaterial researchers designing structures

Analysis and modeling of SDS and DPC micelle SAXS data for membrane protein solution structure characterization

SAXS_DPC_absolute_intensities.zip and SAXS_SDS_absolute_intensities.zip folders contain absolute SAXS intensities in cm-1 of DPC and SDS micelles at different concentrations.Analysed SAXS data are .csv files: "Data IQ over C" represents the absolute intensities normalized to surfactant concentrations for DPC and SDS; "Data I0-Na-RG" represents forward intensity, micelle aggregation number and radius of gyration for each DPC ans SDS concentration, obtained from Guinier analysis of SAXS curves IQ over C; "Data PDDF" corresponds to the pair-distance distribution function of DPC at 5 g.L-1 and SDS at 6.25 g.L-1; "Data Sasview" corresponds to the analytical modeling of the absolute intensities of DPC at 5 g.L-1 and SDS at 6.25 g.L-1.All figures (.png) are those appearing in the Data In Brief article.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV

Polymorphism of urate oxidase in PEG solutions

In a previous paper, we have shown that three different crystal habits exist in the urate oxidase/PEG phase diagram (Vivares, D.; Bonnete, F. J. Phys. Chem. B 2004, 108, 6498-6507). In the present paper, we show with a temperature-controlled device that these different crystal habits correspond to two polymorphs (namely, S/N and P). Furthermore, we point out how an accurate control of the crystallization parameters and a deep understanding of the phase diagram enable us to select one or the other polymorph. Finally, in a narrow part of the phase diagram, we observe an unusual nucleation and growth of one polymorph by epitaxy

Exploring Bovine Pancreatic Trypsin Inhibitor Phase Transitions.

This paper presents an investigation of the phase diagram of BPTI ( bovine pancreatic trypsin inhibitor)/350 mM KSCN at pH 4.9 by direct observation and numerical simulations. We report optical microscopy and light and X-ray scattering experiments coupled with theoretical data analysis using numerical tools. The phase diagram is thoroughly determined, as a function of temperature. Two polymorphs are observed by video microscopy and their solubility measured. In this phase diagram, the liquid-liquid phase separation (LLPS) is metastable with respect to the solid-liquid phase separation. Above the TL-L boundary curve, solutions are composed of a mixture of BPTI monomers and decamers. Attractive interactions are stronger between decamers than between monomers. Below the TL-L boundary curve, the dense phase is highly concentrated in protein and composed of BPTI decamers alone. Thus, the driving force for liquid-liquid or liquid-solid phase separation is the attraction between decamers at low pH. The structure factors of the dense phases are characteristic of repulsive dense phases because of a hard sphere repulsion core, meaning that in the dense phase proteins are actually in contact (interparticle distance of 53 angstrom). In agreement with the Oswald rule of stages, LLPS occurs prior to and impedes the solid nucleation

A preliminary neutron diffraction study of Rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin

Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 angstrom resolution using the LADI instrument from a crystal (grown in D2O) with volume 1.8 mm(3). The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high- resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit- cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 angstrom) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons

Structural studies of urate oxidase using powder diffraction

International audienc