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Growth of Protein Crystals by Syringe-Type Top-Seeded Solution Growth Published as part of the Crystal Growth & Design virtual special issue on the 13th International Conference on the Crystallization of Biological Macromolecules (ICCBM13). Hiroyoshi Matsumura,*,†,‡,^ Keisuke Kakiniuchi,†,‡ Tsutomu Nakamura,§ Shigeru Sugiyama,†,‡ Mihoko Maruyama,†,‡ Hiroaki Adachi,†,‡,^ Kazufumi Takano,†,‡,^ Satoshi Murakami,‡,^,|| Tsuyoshi Inoue,†,‡,^ and Yusuke Mori†,‡,^ †
Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan CREST JST, Suita, Osaka 565-0871, Japan § National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka 563-8577, Japan ^ SOSHO Inc., Osaka 541-0053, Japan Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
)
‡
ABSTRACT: Neutron crystallography is an experimental method for visualizing the hydrogen atoms of proteins, thereby elucidating the mechanism of the target proteins. The technique requires large crystals of proteins to overcome the low flux of the available neutron beam. We previously reported that top-seeded solution growth (TSSG) is an efficient strategy for rapidly growing large protein crystals, but obstacles still remain. Here we report a syringe-type TSSG for growing protein crystals. In this method, a seed crystal is hung in the syringe. The method permits small protein crystals to be attached with the desired crystal orientation under an optical microscope. Similarly to previous results of TSSG, the shape of the lysozyme and superoxide dismutase crystals were strongly influenced by the orientation of the seed crystal. This new approach is expected to expand the scope of target proteins for neutron crystallographic analysis.
1. INTRODUCTION Neutron crystallography provides insights into protein structure protonation details.1,2 This attractive technique provides a better understanding of the enzyme mechanism and supports rational structure-based drug design. However, extremely large crystals of proteins (e.g., >1 to 10 mm3)3 are required in order to obtain an analyzable diffraction pattern from the available neutron beam.4,5 So far, investigators have developed several growth techniques for large protein crystals, including macroseeding, the two-liquid system,6 8 the slow-cooling method,7,9,10 the floating-and-stirring technique (FAST),6,8,9 large-scale hanging drop method,11 and top-seeded solution growth (TSSG).8,12 14 TSSG has been widely utilized to grow inorganic compound crystals, to suppress polycrystallization, and to produce highquality large single crystals. We previously reported that HEWL12 and HIV PR complex crystals8 can be grown by this method, suggesting that TSSG is likely to be generally applicable to many proteins. Intriguingly, the shape of the HEWL crystal is significantly influenced by its seed orientation. It was also confirmed that the silicone glue used in this method did not have a serious impurity effect on crystal growth by laser confocal microscopy combined with differential interference contrast microscopy.15 r 2011 American Chemical Society
While TSSG was certainly an efficient strategy for rapidly obtaining large crystals of proteins, it does have some disadvantages. A major hurdle for this method is the difficulty of attaching the protein crystals with the desired orientation. The seed orientation of the protein crystals is very important because the orientation influences the growth direction of the crystals.8,12 Since the seed crystals cannot be manipulated under an optical microscope, extremely large seed crystals (e.g., >1 mm3) were needed in previous studies.8,12 Another disadvantage is associated with the exchange of the protein precipitant mixture. TSSG is basically the same as the batch method, and one may want to exchange the protein precipitant mixture to carry on the growth. However, the seed crystals are held with a cap in the current device, so it is difficult to exchange the solution. Additionally, the current vessels require a large volume of protein solution, although this depends on the size of the vessels. To overcome these limitations, we report the new development of syringe-type TSSG. Shapable agarose gel was exploited to attach a seed crystal with the desired orientation. The new Received: October 14, 2010 Revised: March 9, 2011 Published: March 16, 2011 1486
dx.doi.org/10.1021/cg101361n | Cryst. Growth Des. 2011, 11, 1486–1492
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Figure 1. (A) Photograph of real setup (left) and schematic illustration (right) of the crystallization setup for syringe-type TSSG. (B) Schematic diagram of the protocol for the attachment procedure. (C) Enlarged illustration of the protocol for attaching the seed crystal with a desired orientation. (D) Photographs of the attachment of the seed crystal. A groove of the agarose gel is shown with dotted lines for clarity.
procedure allows us to attach protein crystals with the desired orientation under an optical microscope. We applied syringetype TSSG to the growth of hen egg white lysozyme (HEWL) with different orientations. Solution stirring was also applied to grow HEWL, resulting in obtaining more bulky crystals. We also applied syringe-type TSSG to the growth of superoxide dismutase from Aeropyrum pernix K1, suggesting the possibility of controlling the growth direction. We discuss the further merits of syringe-type TSSG when the solution is exchanged during repeated macroseeding. We suggest that the new technology may be of general use to grow large protein crystals for neutron protein crystallography.
2. MATERIALS AND METHODS 2.1. Materials. The 1 mL syringe used was purchased from Terumo Co. Ltd. The guide tube was purchased from Hozan Co. Ltd. (Catalogue No. Z-261, extension nozzle). The movable seed holder was a piston of a 50 μL pipette tip of the MICROMAN (Gilson Co. Ltd.). The hen egg white lysozyme (HEWL) was purchased from Seikagaku Co., Japan (Catalogue No. 100940, recrystallized six times). Insoluble and very dense liquid (Fluorinert FC-70) was purchased from Sumitomo 3M Ltd. Silicone glue was purchased from Konishi Co., Ltd., Japan (Catalogue No. 001145). Agarose IX-A (Sigma-Aldrich, Catalog No. A2576) was utilized in this experiment, and the gelling temperature of this agarose is 290 K for 1.5% (w/v) content. 2.2. Protein Expression and Purification of Superoxide Dismutase from Aeropyrum pernix K1 (ApeSOD). PCR was used to amplify the open reading frame of ApeSOD (APE_0741) from genomic DNA of A. pernix K1 (NBRC 100138, provided by National Institute of Technology and Evaluation, Chiba, Japan), and the details of the cloning strategy were described recently.16 The ApeSOD (APE_0741)
was expressed using the vector pET11 (Novagen, Darmstadt, Germany). Escherichia coli Rosetta (DE3) cells harboring the expression vector were cultivated in LB medium containing 0.1 mg/mL ampicillin at 37 C, and protein expression was induced by the addition of 1 mM IPTG. The E. coli cells were disrupted by sonication, and the soluble proteins were subjected to streptomycin and heat treatments, as previously described.17 The resulting solution was dialyzed in 20 mM sodium acetate buffer (pH 4.8) and applied onto a cation exchange column (HiTrap SP, GE Healthcare, Piscataway, NJ, USA). The protein was eluted by a linear gradient of 0 1 M NaCl in the same buffer. The fractions containing ApeSOD were collected, concentrated, and gel-filtered with a Superdex 75 column equilibrated with 20 mM Tris-HCl (pH 8.1) with 150 mM NaCl as the final step. The purified protein was dissolved in 20 mM Tris-HCl (pH 8.1). The protein concentration was determined from its absorbance at 280 nm.18 2.3. Crystallization. The HEWL solution was passed through 0.22 μm filters prior to crystallization. To obtain seed crystals, the HEWL was crystallized at a protein concentration of 50 mg/mL using the batch method in 0.5 M sodium chloride with 0.1 M sodium acetate buffer (pH 4.5) at 293 K. A seed crystal was transferred into the groove of the agarose in a cut pipette tip (Figure 1B) using a nylon loop (Hampton Research), and the seed crystal was adhered to the seed holder with silicone glue (The details will be described in Results and Discussion). After the glue solidified, the seed holder was lifted up with the 0.2 mL of a protein precipitant mixture containing 35 mg/mL HEWL in 0.5 M sodium chloride with 0.1 M sodium acetate buffer (pH 4.5), and then 0.2 mL of Fluorinert was brought into the syringe. To apply solution stirring, a magnetic stirrer bar was placed in the Fluorinert to mildly stir the floating protein precipitant solution (100 rpm). ApeSOD was first crystallized with the hanging-drop vapor diffusion method using a mixture of 3 μL of the protein solution (10 mg/mL in 20 mM Tris-DCl) and 3 μL of precipitant solution containing 20% PEG6000, 8% ethylene glycol, and 100 mM HEPES buffer (pD 7.5). A crystal was transferred and adhered to the seed holder in the same way 1487
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Figure 2. Photographs of the seed crystal of HEWL just after seeding (left) and after 5 10 days (right). The seed crystals were grown at protein concentrations of (A) 20 mg/mL, (B) 50 mg/mL, and (C) 35 mg/mL.
Figure 3. (A) HEWL crystals with (110) seed orientation just after seeding (left) and after 6 days (right), (B) HEWL crystals with (101) seed orientation just after seeding (left) and after 6 days (right), (C) schematic illustration of tetragonal HEWL, with established abbreviations of crystal faces and axes, and (D) plots of increased volume of HEWL seed crystals during crystal growth. The increased volumes of crystals are plotted with the following symbols: blue and red squares, two crystals with (110) seed orientation; and green triangles, two crystals with (101) seed orientation. as HEWL. After the glue solidified, the seed holder was lifted up with the mixture of 0.1 mL of the protein solution (1 mg/mL in 20 mM TrisDCl) and 0.1 mL of precipitant solution containing 20% PEG6000, 8% ethylene glycol, and 100 mM HEPES buffer (pD 7.5), and then 0.2 mL of Fluorinert were brought into the syringe. All crystallization experiments were performed at 293 ( 1.0 K.
3. RESULTS AND DISCUSSION 3.1. Setup of Syringe-Type TSSG. Figure 1A shows the photograph of the real-setup and schematically illustrates the crystallization setup for syringe-type TSSG, an improved version
of TSSG. In this setup, both the seed holder and the syringe plug are movable, and the guide tube is adhered to the syringe with silicone glue. The seed holder passes through the guide tube and penetrates the pore of the syringe plug to access the protein crystals. A thin stick (sculpted polypropyrene pipette tip) is further adhered to the tip of the seed holder to attach smaller seed crystals. The 0.2 mL of Fluorinert was then brought into the syringe. As a result, the protein precipitant mixture was placed onto the Fluorinert. Figure 1B illustrates the protocol for attaching the seed crystal in this method. To obtain seed crystals, hen egg white lysozyme (HEWL) was crystallized at a protein concentration of 50 mg/ 1488
dx.doi.org/10.1021/cg101361n |Cryst. Growth Des. 2011, 11, 1486–1492
Crystal Growth & Design mL using the batch method in 0.51 M sodium chloride with 0.1 M sodium acetate buffer (pH 4.5) at 293 K. A cut pipette tip with grease at one end was attached to the glass slip (Figure 1B, top). The cut tip and an adapter (a cut pipette tip) were jointed together to transfer a seed crystal and inject the protein precipitant mixture using the syringe. The agarose was dissolved and melted in water at a concentration of 2.0% (w/v). The melted agarose was then injected into the cut tip (Figure 1B, top). Before gelation of the agarose, a piston of a 50 μL pipette tip of MICROMAN was put onto the surface of the agarose, and it was kept for 10 min to make a groove in the gellified agarose. After gelation, the protein precipitant mixture was injected into the cut tip using syringe-type TSSG (Figure 1B, second top). A seed crystal was transferred from the hanging drop using nylon loops into the groove of the agarose gel. The orientation of the crystal was adjusted with Micro-Tools (Hampton Research, CA, USA). A seed holder with silicone glue was carefully brought into contact with the seed crystal from the side under an optical microscope (Figure 1B, middle), and the seed holder was subsequently kept in contact with the seed crystal for a few hours to solidify the glue. After the glue solidified, the seed crystal was transferred into the syringe by pulling the seed holder (Figure 1B, next to bottom), and the protein precipitant mixture and was also transferred to the syringe by pulling the syringe plug (Figure 1B, bottom). The 0.2 mL of Fluorinert was then brought into the syringe. The syringe-type TSSG was sealed with the polyethylene cap. As shown in Figure 1C,D, small seed crystals (less than 0.4 mm) can be glued with the desired orientation under an optical microscope with this method, although such attachments cannot be employed in TSSG, as previously reported. 3.2. Optimization of Crystallization Conditions for HEWL. Since the crystallization conditions of HEWL are frequently influenced by the crystallization setup, we first optimized the crystallization conditions of hen egg white lysozyme (HEWL) in
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our setup. The seed crystals were first grown at a protein concentration of 20 mg/mL using the batch method in 0.51 M sodium chloride with 0.1 M sodium acetate buffer (pH 4.5) at 293 K (Figure 2A). No obvious differences in the shape of the seed crystal were observed 10 days later, suggesting that the seed crystal does not grow under these conditions. The seed crystal was then grown under the same conditions but with a protein concentration of 50 mg/mL. As seen in Figure 2B, the higher protein concentration triggered polycrystallization of the seed crystal, although the seed crystal also grew. A seed crystal was subsequently grown under the same conditions but with a protein concentration of 35 mg/mL (Figure 2C). The seed crystal grew larger, and no additional nucleation was observed under these conditions. 3.3. Preference of Crystal Growth by Alternative Seed Orientations. A seed crystal with the desired orientation can be effectively glued under an optical microscope with this method, even though the seed crystals are very small (e.g.,
