Journal of Crystal Growth 232 (2001) 596–602 Crystallization screening directly in electrophoresis gels J.M. Garcı́a-Ruiza,*, A. Hernández-Hernándeza, J. López-Jaramilloa, B. Thomasb a Laboratorio de Estudios Cristalográficos, Institut Andaluz de Ciencias de la Tierra CSIC-Universidad de Granada. Av. Fuentenueva s/n, 18002 Granada, Spain b Center for Microgravity and Materials Research, Research Institute, Room D29. University of Alabama, Huntsville, AL 35806, USA Abstract Screening of crystallization conditions can be directly performed within electrophoretic gels run during the purification steps. The technique is demonstrated for different methods of electrophoretic separations and for two different gels, agarose and polyacrylamide. Two different techniques are proposed to perform the screening: direct diffusive mixing in small gel slices and diffusion technique in gel bands. # 2001 Elsevier Science B.V. All rights reserved. Keywords: A1. Biocrystallization; A1. Gel electrophoresis; A2. Growth from solutions; B1. Biological macro-molecules; B1. Proteins 1. Introduction Electrophoresis is the method currently used by biochemists and molecular biologists to separate biological macromolecules (nucleic acids and proteins) on the basis of size, electric charge, and other physical properties [1]. In practical terms, the molecules under the effect of an electrical current are forced to migrate across a span of gel that works as a molecular sieve. Activated electrodes at either end of the gel provide the driving force. The rate of migration through the electric field depends on the strength of the field, size and shape of the molecules, relative hydrophobicity of the samples, and on the ionic strength and temperature of the buffer in which the molecules are moving. As a result, the separated macromolecules form a number of bands perpendicular to the direction of the electric field [1]. Isoelectric focusing is *Corresponding author. Tel.: +34-958-243360; fax: +34958-243384. E-mail address: [email protected] (J.M. Garcı́a-Ruiz). another electrophoretic technique that works by the formation of a pH gradient across the gel by carrier ampholytes. The ampholytes are mixed in the supplied gel. For instance, in the PhastSystem, the ampholytes are first placed in the electric field to create the pH gradient and then the samples are applied. In the electric field the proteins migrate to their isoelectric points where the overall charge on the protein is neutral. The PhastGels are available without ampholytes so that the range needed can be set by adding the appropriate ampholytes to the gel surface. On other hand, it is also known that gels can be used as growth media to crystallize inorganic and organic compounds [2–5]. The ‘‘gel technique’’ is known to work for thermal gels (for instance, agarose) as well as for chemical gels (silica or polyacrylamide gels). Because native gel electrophoresis will separate protein impurities based on charge and size, and isoelectric focusing will separate impurities with high resolution based on charge differences, we have explored the possibility of utilizing gels from native electrophoresis to screen pH and 0022-0248/01/$ - see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 2 4 8 ( 0 1 ) 0 1 0 8 3 - 1 J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602 precipitating agents for crystallizing abilities. It would save time and protein in the route to obtain crystals. Thus, in a quick and simple way, the protein of interest is purified to remove impurities, which may inhibit crystallization. The purified protein in the gel is then subjected to crystallization by counter diffusion or other techniques. In some cases the need for time consuming chromatographic purification methods may be circumvented. The method is demonstrated to work for two different types of gels, two different crystallization techniques, and two different proteins. 2. Experimental procedure Polyacrylamide and agarose gels were tested because they are commonly used for electrophoresis. Hen egg white lysozyme (pI=11.4) and horse spleen ferritin (pI=4.3) were chosen as test proteins because of these pI values. Two different crystallization methods were designed and are described below. Native horizontal electrophoresis in agarose gels was carried out with a submarine electrophoresis system purchased from BIO-RAD. The gel was made of Agarose l (code 17-0424-02) from Pharmacia Biotech, at concentration 0.5 and 1% w/v. Ferritin in the gel was buffered to pH 7.5 with 200 mM Tris-HCl solution. Six 20 ml lanes of 25 mg/ml horse spleen ferritin (Fluka-4503 lot 125H70402) were loaded. For lysozyme (Fluka62971 (lot: 331429/1595), five or six 40 ml lanes at concentration ranging from 100 to 200 mg/ml were tested. The applied voltage was 100 V for 60 min for ferritin and 100 V for 120 min for lysozyme. Native electrophoresis in 7.5% polyacrylamide gels was carried out with a BIO-RAD MiniProtean II. The gel was buffered to pH 8.3 with 250 mM Tris-glycine solution. Ten 10 ml lanes of 12.5 mg/ml horse spleen ferritin were performed. A voltage of 150 V was applied for 75 min. For isoelectric focusing, the procedure was also a classical one. A single lane sample applicator (normally used for 2D gels) was used to apply 100 mg/ml ferritin (Sigma Chemical Co. St. Louis, MO, USA) to an Amersham Pharmacia Biotech 597 (Piscataway, NJ, USA) 3-9 pI isoelectric focusing PhastGel. Since the ampholytes act as buffers, the precipitating agent was dissolved in a concentrated buffer (0.2 M) to adjust the pH to 5.0. Two different screening methods are used after separation is achieved: (a) Gel slices: It consists of soaking small pieces (2 2 mm) of gel containing the protein bands into the precipitating agent solution. This screening method is similar to a batch method. However, unlike batch where precipitating agent and protein are directly mixed, this method allows the precipitant to diffuse into the gel slice. Because the gel layer is thin, diffusive mixing occurs quickly. After mixing occurs, the band of gel containing the protein is cut in small pieces. To precipitate the protein, various concentrations of precipitating agents previously buffered at the desired pH are poured onto each slice of gel. Cadmium sulfate at concentration ranging from 2 to 10% (w/v) buffered at pH=5.3 with 200 mM sodium acetate were used for ferritin experiments. Sodium chloride at concentrations of 13–30% (w/v) buffered to pH=4.5 with 50 mM sodium acetate were used for lysozyme experiments. (b) Whole band: This screen is actually a counterdiffusion experiment. It consists of using the whole band of gel containing the protein and soaking one of its sides perpendicular to the protein bands in the solution of the precipitating agent. The precipitating agent diffuses into the gel producing a gradient from high to low supersaturation. Because of their flexible plastic backing, PhastGels can be inserted into a solution of precipitating agent to a depth of 0.5 cm. For native electrophoresis, the whole band of gel containing the protein is oriented in the precipitating agent so that the protein band is perpendicular to the precipitating agent, allowing it to diffuse along the protein band. Cadmium sulfate (10% w/v in 200 mM sodium acetate at pH=5.0) was used to precipitate ferritin while 30% (w/v) NaCl in 50 mM sodium acetate at pH=4.5 was used to precipitate lysozyme. 598 J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602 3. Results and discussion The use of native horizontal electrophoresis is demonstrated using 100 mg/ml lysozyme and 1% agarose. Fig. 1 shows precipitates obtained at different concentrations of NaCl using gel slices. Crystals appear at 15% NaCl, while a higher NaCl concentration yields spherulites and sheaf-ofwheat morphologies characteristic of high supersaturation. The use of the whole band with counterdiffusion method is illustrated by a crystallization screening inside electrophoretic gels of agarose Fig. 1. Lysozyme precipitates obtained at different concentration of NaCl. From top to bottom, 30, 20 and 15% (w/v). containing isolated bands of ferritin (Fig. 2). The precipitating agent (CdSO4) diffuses from the bottom. The screening is performed therefore from high supersaturation (lower part) to low supersaturation (upper part). Observations confirmed what is expected from the counter-diffusion technique. Thus, in the lower part fractal dendrites are observed, followed by a region of spherulites, later a region of dendritic crystals and finally a region where faceted octahedral crystals form. This type of screening permits scanning of a wide range of concentrations for a given precipitating agent and a given pH value. Note for instance in the above results that crystal dendrites appear in a wide range of crystallization conditions, while the range for octahedral crystals is very narrow. This is in agreement with the precipitation behavior of ferritin in presence of CdSO4 concentration obtained in a separate work [6]. This precipitation behavior of ferritin was confirmed by the technique of direct mixing of precipitating agent with gel slices containing the protein. Fig. 3 shows the results obtained as a function of the CdSO4 concentration. Spherulites are obtained at a concentration of 10% (w/v). Dendritic forms occur at 4 at 8% CdSO4 while crystals become faceted at 4%. Octahedral crystals appear at 2% CdSO4. The technique also works for those cases in which the protein is no longer pure because of polymerization, denaturation or just contamination with foreign compounds. To illustrate this, we present the results obtained by using polyacrylamide electrophoretic gels. As shown in Fig. 4, three protein bands were separated. The broad and denser one corresponds to the monomeric form of ferritin, as checked by light scattering in a separate experiment, while the narrow and lighter ones correspond to the dimer and trimer. Gel slices were cut from the three bands and drops of CdSO4 at different concentrations were poured on them. After 24 h, faceted crystals of ferritin (Fig. 4) appear in the gel slices soaked in 10 and 8% CdSO4 and they appear only on the gel slices of the band containing monomeric ferritin. After one month, crystals did not appear in the gel slices containing either the dimeric or the trimeric ferritin forms. Note also that the shift of the J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602 599 Fig. 2. Counter-diffusion screening performed within a 1% agarose electrophoretic gel containing a band of monomeric ferritin. precipitation behavior to higher precipitating agent concentration is due to the lower concentration of protein used in the electrophoretic run. These results agree with those described by Thomas et al. [7] for the iron free ferritin (apoferritin). The pore size of agarose gels is very large for most of the proteins and they are normally used 600 J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602 Fig. 3. Direct mixing screening using slices of a 1% agarose electrophoretic gel containing monomeric ferritin. The CdSO4 concentration (%w/v) varies as labeled in the figure. Fig. 4. Above: a native PAGE of horse spleen ferritin. From upper to lower, the protein bands correspond to the trimeric, dimeric and monomeric forms. Below: examples of ferritin crystals obtained from slices of gels containing the monomeric band. for electrophoresis of nucleic acids. Therefore, when agarose gels at a concentration of 1% are used to electrophoretically separate a protein like lysozyme (molecular weight of 14 kDa), 2D screening takes place when the whole band is screened with the counter-diffusion arrangement. In the absence of an electric field, when the counterdiffusion screening is set, the precipitant diffuses across the band and, concurrently, the protein also diffuses sideways broadening the band. The combination of both diffusion trends results in a two-dimensional V-shaped pattern which results in a range of protein concentrations as shown in Fig. 5. Thus, protein concentration can also be screened. We obviously use chemical agents known to precipitate the tested protein in this practical demonstration of the technique. In a test for a new protein, one should use the whole set of the preferred screening kit in the gel slice technique. About fifty 1.5 1.5 mm2 gel slices can be obtained from a 8 cm long and 0.3 cm wide band. Therefore, 50 different chemical variations to precipitate the protein can be tested. We recommend to use this technique to screen pH and type of precipitating agent. All the precipitating agents currently used in crystallization screening should be compatible with the gel slice technique because, J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602 601 Fig. 5. Screening pattern obtained when the pore size of the gel (0.5% agarose gel) is much larger than the protein size (HEW lysozyme). Precipitating agent is 30% w/v NaCl. even if these chemicals cannot diffuse into the gel, the protein diffuses from the gel into the precipitant solution. the protein is crystallized just after its separation. In short, the corollary of this work is: do not discard your gels after electrophoresis, use them to search for potential crystallization conditions. 4. Conclusion We demonstrate that different electrophoresis gels can be directly used for screening of crystallization conditions after being used to analyze the protein during the purification steps. The use of this method drastically reduces the waste of protein in further screening as it uses a material that is often discarded after use in the previous and unavoidable process of purification. In addition, for those proteins that have a strong tendency to denature or to aggregate, this method represents an advantage because Acknowledgements This research has been supported by the Spanish Ministerio de Educación y Ciencia, Consejo Superior de Investigaciones Cientificas and European Space Agency. J.M. Garcı́a-Ruiz also acknowledge the University Space and Research Association for a stay at the Marshall Space Flight Center. The authors acknowledge the improvement of the clarity of the manuscript by an anonymous referee. 602 J.M. Garcı´a-Ruiz et al. / Journal of Crystal Growth 232 (2001) 596–602 References [1] B.D. Hames, D. Rickwood, Gel Electrophoresis of Proteins, A Practical Approach. Oxford University Press, Oxford, 1990. [2] H.K. Henisch, Crystal Growth in gels and Liesegang rings, Cambridge University Press, Cambridge, 1988. [3] J.M. Garcı́a-Ruiz, Key Eng. Mater. 58 (1991) 87. [4] M.C. Robert, J.M. Garcı́a-Ruiz, O. Vidal, F. Otálora, in: A. Ducruix, R. Giegé (Eds.), Crystallization of Nucleic Acids and Proteins: A Practical Approach, IRL Press, Oxford, 1999, pp. 149–177. [5] F.J. Rotella, C.S. Giometti, X-ray Diffraction From Proteins Crystallized in Polyacrylamide Gels. Abstracts’ Book of the 1999 ACA Meeting, 1999. [6] B.R. Thomas, J.M. Garcı́a-Ruiz, unpublished. [7] B.R. Thomas, D. Carter, F. Rosenberger, J. Crystal Growth 187 (1998) 499.
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