Effect of macromolecular impurities on lysozyme solubility and crystallizability: dynamic light scattering, phase diagram, and crystal growth studies

Skouri, M.; Lorber, Bernard; Giegé, Richard; Munch, J. P.; Candau, Jean

doi:10.1016/0022-0248(95)00051-8

Cited by 93 publications

(73 citation statements)

References 39 publications

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“…Many light scattering studies have addressed the behavior of protein solutions under crystallizing conditions in terms of aggregate formation and nucleation induction time [2][3][4]. These studies relate solution conditions to the formation of nuclei and the time required for the first appearance of these nuclei, the induction time.…”

Section: Introductionmentioning

confidence: 99%

Dependence of nucleation kinetics and crystal morphology of a model protein system on ionic strength

Bhamidi

Skrzypczak‐Jankun

Schall

2001

Journal of Crystal Growth

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Nucleation rate data for hen egg-white lysozyme crystallization were obtained using a particle counter. Tetragonal lysozyme crystals were expected to form at the temperature and solution conditions of these experiments: 48C, pH 4.5 with 0.1 M sodium acetate buffer and 2-6% NaCl (w/v). The rates varied as expected, as smooth monotonic functions of supersaturation at 2%, 3% and 6% NaCl. However, at 5% NaCl, a great deal of scatter in the data was observed. At 2% and 3% NaCl, all the batches contained crystals with tetragonal morphology. At 6% NaCl, almost all of the vials contained the white powder with few or no tetragonal crystals. At 5% NaCl concentration, a mixture of tetragonal crystals and powder formed in varying proportions in all the vials as observed by visual inspection. The powdery material was examined using optical microscopy and was seen to consist of needles with regular structure and sharp, faceted edges. Powder diffraction data from these needles was inconsistent with experimental powder diffraction data from tetragonal lysozyme crystals. It is possible that at high salt and protein concentrations liquid-liquid separation occurred and yielded a crystal polymorph. #

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Section: Introductionmentioning

confidence: 99%

Dependence of nucleation kinetics and crystal morphology of a model protein system on ionic strength

Bhamidi

Skrzypczak‐Jankun

Schall

2001

Journal of Crystal Growth

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“…[13][14] However, they may interfere with the nucleation process and may cause increased twinning 15 and loss of faceted faces. 13,16 Impurities structurally similar to the macromolecule of interest are more likely to be incorporated into the crystal in significant quantities. [15][16][17] This may result in significant changes in crystal morphology due to slower growth on one crystal face relative to another, which results in a shortening 17,18 or lengthening of the crystal 19 in comparison to those grown in pure solutions.…”

Section: Introductionmentioning

confidence: 99%

Investigating the Effect of Impurities on Macromolecule Crystal Growth in Microgravity

Snell

Judge

Crawford

et al. 2001

Crystal Growth & Design

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Chicken egg-white lysozyme (CEWL) crystals were grown in microgravity and on the ground in the presence of various amounts of a naturally occurring lysozyme dimer impurity. No significant favorable differences in impurity incorporation between microgravity and ground crystal samples were observed. At low impurity concentration the microgravity crystals preferentially incorporated the dimer. The presence of the dimer in the crystallization solutions in microgravity reduced crystal size, increased mosaicity, and reduced the signal-to-noise ratio of the X-ray data. Microgravity samples proved more sensitive to impurity. Accurate indexing of the reflections proved critical to the X-ray analysis. The largest crystals with the best X-ray diffraction properties were grown from pure solution in microgravity.

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“…Furthermore, they can cause dislocation and cracks, the formation of soluble aggregates, and the degradation of crystal mosaicism (Caylor et al 1999). Studies on lysozyme using light scattering have shown that impurities could associate with lysozyme, promoting the formation of ill-shaped and twinned crystals (Lorber et al 1993, Skouri et al 1995. Because crystal formation is sensitive to many factors, crystallisation of a protein can be the rate-determining step in protein structure determination using X-ray crystallography.…”

Section: Resultsmentioning

confidence: 99%

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

Anggraeni¹

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A promising alternative to replace the current egg-or cell culture-based technology for vaccine production from live viruses is virus-like particle (VLP) technology based on a microbial platform. VLPs are macromolecular assemblies of viral capsid proteins, which have been shown to tolerate insertion of antigen modules via genetic recombinant technology, yielding modular VLPs. Many studies on modular VLPs presume that when a peptide antigen element is taken out from the intact proteins and then modularised on VLPs, it is unable to fold into its native structure. However, until now, presentation of a peptide antigen element on a VLP and the impact of the display strategy to present the antigen element on the quality of the resulting antibodies (i.e. the ability of the antibodies to recognise the intact protein) are not fully understood. This thesis aims to understand the underlying fundamentals regarding modularisation of peptide antigen elements on VLPs for induction of high-quality antibodies. A hypervariable receptor-binding domain, Helix 190 (H190), from the hemagglutinin protein of influenza A virus was used as a model for modularisation on VLPs from murine polyomavirus (MuPyV) VP1 protein. Four major findings are presented. Firstly, two display strategies, i.e. arraying of H190 in tandem repeats and the use of helix promoter elements, were shown to display H190 in its immunogenic form equally. However, modularisation using tandem repeat display induced antibodies of a higher quality than modularisation using helix promoter elements.Secondly, the quality of antibodies induced by the tandem repeat display bearing two copies of H190 was optimum, thus no significant improvement was observed following the use of adjuvant or increasing the copy number of H190. Additionally, the increase in the copy number of H190 was shown to reduce the assembly capability and solubility of modular VP1 in an environment that was optimised for wild-type VP1. Thirdly, this thesis shows the novel finding in the use of flanking ionic elements to stabilise VLP precursors, termed as capsomeres, bearing two copies of H190 containing a hydrophobic stretch, which caused aggregation. Fourthly, the first steps towards obtaining the atomic crystal structure of presented H190 on a modular protein were performed, i.e. a mild and satisfactory laboratory process was developed to achieve high-purity modular VP1 capsomeres, unattainable using previously established expression and purification process of wild-type MuPyV VP1. This thesis shows a step forward towards understanding the presentation of a peptide antigen element on a VLP that enables induction of highquality antibodies, and towards VLP engineering to manipulate the aggregation and solubility of modular VP1. VLP technology based on a microbial platform presented here is iii a potentially safe and effective alternative vaccine candidate that targets a hypervariable peptide antigen element. The speed of the microbial platform allows a rapid response to the hypervariability of the pe...

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Effect of macromolecular impurities on lysozyme solubility and crystallizability: dynamic light scattering, phase diagram, and crystal growth studies

Cited by 93 publications

References 39 publications

Dependence of nucleation kinetics and crystal morphology of a model protein system on ionic strength

Dependence of nucleation kinetics and crystal morphology of a model protein system on ionic strength

Investigating the Effect of Impurities on Macromolecule Crystal Growth in Microgravity

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

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