Solvent freeze out crystallization of lysozyme from a lysozyme‐ovalbumin mixture

Hen egg white lysozyme (HEWL) crystallization conditions from an ovalbumin‐lysozyme mixture were found by screening tests and further located in pseudo‐phase diagrams. This information was used to set up the initial conditions for the solvent freeze out (SFO) process. The process uses the freezing of ice to create the supersaturation for the proteins to crystallize out of the solution. The crystallization of HEWL (15 mg/mL) out of a lysozyme‐ovalbumin mixture (1.7 mg/mL) is carried out by SFO. Under the reported conditions, a crystallization yield of 69 % was obtained. A mean crystal size of 77.8 µm was enhanced in a crystallization time of 15.1 h. The lysozyme nature of the crystals is proven by SDS PAGE and enzymatic activity tests. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Small-angle X-ray scattering study of conditions for the formation of growth units of protein crystals in lysozyme solutions

The structural composition of lysozyme solutions favorable for the formation of the tetragonal form of protein crystals was studied by synchrotron-based small-angle X-ray scattering depending on the protein concentration and the temperature. Along with lysozyme monomers, dimers and octamers are found in crystallization solutions; the octamer content increases with an increase in the protein concentration.     

Effects of purification on the crystallization of lysozyme

We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20°C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal ↔ orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.     

Small-angle X-ray scattering study of the influence of solvent replacement (from H<Subscript>2</Subscript>O to D<Subscript>2</Subscript>O) on the initial crystallization stage of tetragonal lysozyme

The composition of lysozyme solutions in D₂O under conditions favorable for the formation of tetragonal crystals has been investigated at different protein concentrations by small-angle X-ray scattering using the synchrotron radiation. In addition to lysozyme monomers, dimeric and octameric species are found in the crystallization solutions; the octamer content increases with an increase in the protein concentration. A comparison of the data with those obtained under similar conditions but with H₂O used as a solvent has shown that the replacement of light water with heavy one leads to increase of octamer volume fraction in solution.     

A more clear insight of the lysozyme crystal composition

Crystallization can be used as a purification method for proteins. Lysozyme was chosen as a model substance. Changing crystallization conditions will lead as shown to different lysozyme crystal morphologies with different properties. Beside others, lysozyme crystals can show a Tetragonal, High Temperature and Low Temperature Orthorhombic crystal morphology. Experiments such as conductivity measurements, pH tests, chloride detection tests, experiments using methylene blue as a dye and dissolution experiments were carried out to investigate the composition of the lysozyme crystals. It is proven that lysozyme crystals are made up of the initial buffer solution components: lysozyme (the protein), water which is part of the crystal lattice, salt ions which are attached to the protein molecule and voids filled with the buffer solution containing the crystallization agent (e.g. salt). Interesting dissolution behaviours of the lysozyme crystals were observed which are not described so far elsewhere (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Deuterated hen egg-white lysozyme crystals: Optimization of the growth conditions and morphology

Deuterated and protonated tetragonal lysozyme crystals are grown using the hanging-drop vapor-diffusion method. The size of the lysozyme crystals grown is determined as a function of the concentration of sodium chloride used as a precipitant. It is found that crystallization leads to the formation of lysozyme crystals with three different habits. Morphological and X-ray diffraction analyses of the deuterated and protonated lysozyme crystals demonstrate that, despite the different habits, all the crystals grown belong to the tetragonal crystal system. The simple forms of lysozyme crystals are revealed. It is shown that the habits of the lysozyme crystals are determined by the specific combinations of simple forms. The mechanisms responsible for the formation of lysozyme crystals with different habits are discussed.     

Implementation of Temperature-Controlled Method of Protein Crystallization in Microgravity

An experimental scientific equipment for implementing temperature-controlled protein crystallization in capillaries under microgravity has been developed, fabricated, and tested. This crystallization method, providing on-line separate control of crystal growth both in the stage of nucleation of crystals and during their further growth, requires small amounts of protein solution. The equipment has been tested on board of Foton-M4 spacecraft (growth of lysozyme protein crystals of high structural quality in microgravity) using a cyclogram developed in ground-based experiments. The results obtained have demonstrated efficiency and importance of the developed equipment and method for growing biomacromolecular crystals of high-structural quality.

     

The dissolution phenomenon of lysozyme crystals

Dissolution studies on lysozyme crystals were carried out since the observed dissolution pattern look different from non‐protein dissolved crystals. The Tetragonal, High Temperature and Low Temperature Orthorhombic morphologies, crystallized using sodium chloride, were chosen and the influence of different pH, salt and protein concentration on their dissolution was investigated. An increase in pH and/or salt concentration can modify the dissolution behaviour. The pattern of the crystals during the dissolution process will, therefore, develop differently. Frequently a skeleton like crystal pattern followed by a falling apart of the crystals is observed. The multi‐component character of the lysozyme crystal (protein, water, buffer, salt) as well as “solvatomorphism” gives first insights in the phenomena happening in the dissolution process. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Crystallization and stability of different protein crystal modifications: A case study of lysozyme

The crystallization, including both the phase diagram and the phase transition of hen egg white lysozyme (HEWL) was investigated. A tetragonal modification and a needle modification were obtained during crystallization. The phase diagram and stability of two modifications in both acid and basic pH solutions (pH 4.5, 8.0 and 9.0) were determined. Besides in acid solutions, the well‐known tetragonal crystals can also be obtained in basic solutions at low temperature (7 °C) while the needle like modification can only crystallize in a basic solution. Based on the phase diagram, phase transfer behavior was found to exist between the two modifications. In basic solutions, tetragonal modification can transfer to needle shaped crystals. This process can be affected by a changing of pH and temperature. While in acid buffer, the needle shaped crystals dissolve and tetragonal crystals crystallize and remain in solution.     

Crystallization of proteins under an external electric field

An external electric field affects the crystallization of proteins when applied under some conditions of temperature, pH, and precipitating agent composition. As suggested in the theoretical part of this paper, it produces large protein concentration gradients inside the mother liquor leading to a local supersaturation area in the crystallization solution. Such an experiment has been used for the first time on the crystallization of a protein. The effects of an external electric field on the crystallization of hen egg-white lysozyme at 293 K, pH 4.5, and two NaCl concentrations (0.6–0.7 M) have been investigated using the vapor diffusion method. The application of electric field results in a smaller number of crystals with larger size. The crystals grew at the droplet surface, near the cathode. The nucleation rate is drastically reduced and this experimental method could be used to control the number of crystals in the droplet.     

Sedimentation as a tool for crystallization from protein mixtures

Crystals from apoferritin which is an iron‐free form of protein ferritin were obtained from protein mixtures lysozyme/apoferritin using sedimentation under high gravity. Solution containing apoferritin at concentration as high as 5mg/ml in the presence of 25mg/ml lysozyme and overlaid on 5%(w/v) CdSO₄ in 0,2M/L NaAC, pH=5 still favors apoferritin crystal formation under normal gravity conditions, but at apoferritin concentrations <0,5mg/ml (∼1,14µM/L) in 25mg/ml (∼1,71mM/L) lysozyme only the sedimentation in a centrifuge appears to be useful for separating the apoferritin molecules from the mixture followed by apoferritin crystallization in the same system. The very high molecule number ratio (∼1:10³) of two proteins is used to stress on the observed effect. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Solubility of tetragonal and orthorhombic lysozyme crystals under high pressure

Two-beam interferometry was applied to measure lysozyme solubility under high pressure. This rapid method allowed determination of one data point within 3 h. The solubility of tetragonal lysozyme crystals was determined as a function of temperature at 0.1, 50, and 100 MPa, and that of orthorhombic crystals was measured at 0.1 and 100 MPa. The solubility of tetragonal crystals increased with pressure; however, that of orthorhombic crystals decreased. In both cases, the enthalpy and entropy of dissolution decreased with pressure.     

The formation of stereocomplex in asymmetric blends of PLLGA and PDLA and the effect of stereocomplex on PLLGA crystallization

The isothermal and non‐isothermal crystallization behaviors of asymmetric poly(L‐ lactic acid‐ co‐ glycolic acid) (PLLGA)/poly(D‐lactic acid) (PDLA) blends was investigated at PDLA loadings of up to 10 wt%. A simple solvent‐casting procedure, was developed for the preparation of PLLGA/PDLA stereocomplex crystallites dispersed in a PLLGA matrix. The formation of stereocomplexes in the PLLGA matrix was verified by differential scanning calorimetry (DSC) and X‐ray diffraction (XRD). Non‐isothermal crystallization measurement demonstrated that the addition of PDLA significantly accelerates the crystallization of PLLGA. Subsequent assessment of the influence of PDLA content on the isothermal crystallization kinetics of PLLGA by the Avrami equation confirmed that the crystallization rate of PLLGA is enhanced by the addition of PDLA contents, with a reduction in the half crystallization time (t_1/2) and increase in the crystallization rate constant (k) values. A PDLA content of 1–7 wt% was found to particularly enhance the crystallization rate of PLLGA, from which it is inferred that the PLLGA/PDLA stereocomplex crystals act as effective nucleus to enhance the crystallization of PLLGA.     

Protein crystallization in low gravity by step gradient diffusion method.

Two-step crystallization experiments were conducted in low gravity employing a liquid-liquid diffusion method in an effort to eliminate problems associated with protein crystal growth under the supersaturating conditions required for nucleation. Experiments were performed in diffusion cells formed by the sliding of blocks on orbit. Step gradient diffusion experiments consisted of first exposing protein solutions in diffusion half-wells for brief periods to initiating buffer solutions of high precipitant concentrations to induce nucleation followed by expoure of the same protein solutions to solutions of lower precepitant concentration to promote growth of induced nuclei into crystals. To avoid convective disturbances that occur when solutions of discrepant densities are interfaced at normal gravity, crystallization of hen egg-white lysozyme and rabbit skeletal muscle aldolase by step gradient diffusion was investigated in low gravity on four NASA space shuttle flights. In general, the largest ctystals of both proteins formed at the highest initiating precipitant concentration used, which is consistent with nuclei formation upon brief exposure to high precipitant concentration, and that these nuclei are competent for sustained growth at lower precipitant concentration. The two-step approach dissociates nucleation events from crystal growth allowing parameters affecting nucleation kinetics such as time, precipitant concentration and temperature of nucleation to be varied separately from conditions used for post-nucleation growth.     

Isothermal crystallization behavior and crystal structure of poly(ethylene terephthalate‐co‐1,4‐cyclohexylene dimethylene terephthalate) (P(ET/CT)) copolyesters

Poly(ethylene terephthalate) (PET) and a series of poly(ethylene terephthalate‐co‐1,4‐cyclohexylene dimethylene terephthalate) (P(ET/CT)) copolyesters with different molar ratios of ethylene glycol (EG) to 1,4‐cyclohexanedimethanol (1,4‐CHDM) were investigated by their isothermal crystallization behavior, transparency, crystal structure and morphology changes in different isothermal crystallization process using differential scanning calorimetry (DSC), digital photos, wide‐angle X‐ray diffraction (WAXD) and polarized optical microscopy (POM). The results revealed that P(ET/CT) copolyesters with ≤ 15 mol% 1,4‐CHDM and ≥ 50 mol% 1,4‐CHDM, such as P(ET/CT)(85/15), P(ET/CT)(50/50) and P(ET/CT)(30/70), were crystallizable, while that with 30 mol% 1,4‐CHDM, namely P(ET/CT)(70/30), was amorphous. The crystallization rate, crystallinity and transparency of these copolyesters underwent the same isothermal crystallization process, i.e. it first decreased and then increased remarkably with the increase of 1,4‐CHDM content. Accordingly, the crystal structure of these copolyesters changed from PET‐type lattice to PCT‐type lattice when the copolyester with around 30 mol% 1,4‐cyclohexylene dimethylene terephthalate (CT) unit. Interestingly, the small incorporation of 1,4‐CHDM into PET could lead to the formation of larger spherulite crystals than that of PET. But small and grainy crystallites appeared with further increase in the 1,4‐CHDM content.     

In situ monitoring and control of lysozyme concentration during crystallization in a hanging drop

Fiber optic Raman spectroscopy combined with a partial least-squares regression model was demonstrated as a monitor of lysozyme concentration during crystallization in a hanging drop experiment in real time. Raman spectral features of the buffer and protein were employed to build the regression model. The use of fiber optic technology coupled with Raman spectroscopy, which is ideal for use with aqueous solutions, results in a powerful noninvasive probe of the changing environment within the solution. Lysozyme concentrations were monitored in experiments at a constant reservoir ionic strength. Data from these uncontrolled experiments were used to determine rates of supersaturation, induction times, and the number and size of the resultant lysozyme crystals. Control experiments were performed by introducing step changes in the reservoir ionic strength. The step changes were initiated by comparing in situ rates of supersaturation with the rates of supersaturation calculated from the uncontrolled data. Monitoring the concentration changes of the lysozyme within the hanging drop permits a measurement of the level of supersaturation of the system and enhances the possibility of dynamic control of the crystallization process.     

The crystallization of lysozyme in the system of ionic liquid [BMIm][BF4]‐water

Lysozyme crystallization was conducted in the ionic liquid (IL) 1‐butyl‐3‐methylimidizolium tetrafluoroborate ([BMIm][BF₄]) with different buffer/IL proportions. It was found that the addition of [BMIm][BF₄] could promote the crystallization process, during which more lager single crystals with controllable morphologies could be obtained due to the manageable crystal growth velocity. A probable explanation was proposed based on the influence of the ionic polarization and kinetics in the lysozyme crystallization. Moreover, the transform in coordination number and the relative growth rate of different crystal faces were discussed. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The effect of diluting crystallization droplets on protein crystallization in vapor diffusion method

In this study, effects of diluting either protein or crystallization agents in the droplets on the success rate of protein crystallization was investigated. Diluting the crystallization agent was found to increase the success rate of protein crystallization. Theoretical analysis showed that, concentration ranges of both protein and crystallization agent that can be scanned during the vapor diffusion process are wider with diluting the crystallization agent than that without dilution, resulting in more opportunities for the crystallization solution to be in the nucleation zone. On the other hand, diluting protein could lead to controversial results depending on the location of the initial concentration relative to that of the nucleation zone in the phase diagram. The method of diluting the crystallization agent is therefore proposed as an alternative modification to the conventional vapor diffusion method for obtaining more crystallization conditions in protein crystallization screening. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Mechanism of aggregation and intergrowth of crystals during bulk crystallization from solutions

Available literature data on aggregation kinetics of crystals of a number of salts during their bulk crystallization from solutions have been analysed. The proposed earlier mechanism of aggregation and intergrowth of crystals during bulk crystallization owing to formation of nucleus‐bridges between crystals was tested and confirmed. The aggregation kinetics of crystals was described by the familiar Smoluchowski equation for coagulation of colloidal particles. However, in a bulk crystallization process, the aggregation constant in this equation decreased as supersaturation in a solution lowered. An expression for the aggregation constant in this equation was proposed. The proposed mechanism of crystal intergrowth duringt bulk crystallization allowed evaluating the specific surface energy of tested salts, which turned out to be in reasonable agreement with published literature data. It was concluded that the intergrowth of crystals during bulk crystallization from solutions proceeded via formation of nucleus‐bridges between crystals. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Study of lactose crystallization in water‐acetone solutions

This paper describes a study on the process of lactose crystallization using a water‐acetone solution. The selection of lactose was based on its significance for the pharmaceutical and food industries and on the fact that the crystallization of this organic compound has been little studied and is, unlike inorganic compounds, complex. The objective was to achieve lactose batch crystallization of solutions by analyzing the crystal growth under different operating conditions. To determine solubility curves, the experiments were carried out based on gravimetric methods. All the crystallization experiments were performed according to the methodology proposed by Nývlt in 1985, who uses the temperature at which the first crystals appear (nucleation) to establish the width of the metastable zone and the induction time. The results showed that crystals with different average diameters, shape factors, and recovered mass were obtained for different water‐acetone compositions. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)