A New Form of Crystalline Rubisco and the Conversion to Its Common Dodecahedral Form Prachuab Kwanyuen,*,† Sandra M. Allina,‡ Arthur K. Weissinger,‡ and Richard F. Wilson† United States Department of Agriculture, Agricultural Research Service and Crop Science Department, North Carolina State University, Raleigh, North Carolina 27695-7631 Received July 10, 2002
Abstract: In this paper, we present a new purification procedure that yields a new crystalline form of rubisco and has enabled us to completely remove this most abundant protein from tobacco leaf extract. The crystals formed within 48 h after refrigeration at 4 °C at pH 5.6. However, these crystals were not well-ordered crystals and lacked well-defined facets or edges. The remaining leaf extract (fraction 2 protein) was void of rubisco. Conversion of this new crystalline form of rubisco to its common dodecahedral form was achieved by dialysing the protein solution in Tris buffer at pH 8.0 or purified water. Since the molecular size of its large subunit of rubisco (55 kD) is similar to that of the papillomavirus capsid protein, L1 (57 kD), its complete removal from fraction 2-protein may facilitate the detection, purification, and recovery of the L1 protein. Keywords: rubisco • purification • crystallization • recrystallization • dodecahedron • fraction 2-protein
Introduction Research and development of proteins and biological compounds for medicinal uses via biotechnology using transgenic plants such as tobacco have been in progress for over a decade. One example is the production of immunoglobulins and functional antibodies in transgenic tobacco.1 Presently, an intensive collaborative effort is underway to develop an economical method for producing a prophylactic vaccine against HPV-16, a papillomavirus known to cause cervical cancer. One approach is to express the papillomavirus capsid protein, L1, in transgenic tobacco. However, tobacco and other C3 plants are abundant in rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase, E.C. 4.1.1.39), an enzyme of photosynthetic CO2 assimilation that comprises of 50-60% of total soluble protein in the leaf extract. In addition, the large protein subunit of rubisco (55 kDa) is similar in mass to the L1 protein (57 kDa). Thus, the great abundance of rubisco in leaf extract impedes the success in detection, purification, and recovery of the L1 protein. Therefore, removal of most if not all rubisco from leaf extracts of transgenic tobacco or other plants used for this * To whom correspondence should be addressed. Phone: (919) 513-1479. Fax: (919) 856-4598. E-mail:
[email protected]. † United States Department of Agriculture. ‡ North Carolina State University. 10.1021/pr025548e CCC: $22.00
2002 American Chemical Society
purpose is necessary to facilitate the production, recovery, and purification of this protein to make the vaccine. Purification and crystallization of rubisco in its dodecahedral form from tobacco leaf extract have been reported.2,3 The techniques require gel filtration chromatography with G-25 or G-50 Sephadex. This purification step is time-consuming and generates a large amount of liquid. Although a large quantity of the dodecahedral form of crystalline rubisco was isolated, a majority of the rubisco still remained soluble in the leaf extract. In this paper, we present an improved process for removal of rubisco from tobacco leaf extract and its recrystallization. The ease of conversion of this new crystalline form to its dodecahedral form of rubisco will also be described.
Materials and Methods Plant Material. Tobacco leaves (Nicotiana tabacum L., cv. KY 14) were obtained from plants grown in a greenhouse at North Carolina State University. All procedures were performed at room temperature unless otherwise indicated. Purification and Crystallization of the Dodecahedral Form of Rubisco. Crude leaf extract was obtained by homogenizing fresh tobacco leaves in a Waring blender at a ratio of 2:1 (w/v) with 2% sodium meta-bisulfite (Na2S2O5) solution. The homogenate was filtered through several layers of cheesecloth. The purification procedures of Chan et al.2 and Lowe3 were followed with the following modifications to obtain an original dodecahedral form of rubisco for comparison and as standard. The final concentration of Tris-HCl in the crude leaf extract was 25 mM; and the pH was adjusted to 8.0 with 1 N NaOH. The extract was centrifuged at 30000g for 30 min. The supernatant was passed through a Sephadex G-25 column equilibrated with 25 mM Tris-HCl, pH 8.0. The ratio of sample to column bed volume was 1:10 (v/v). The protein fraction eluted in the void volume was collected and placed in a refrigerator for 48-72 h for crystallization of the dodecahedral form of rubisco. Recrystallization. The dodecahedral crystalline form of rubisco was collected by centrifugation at 3000g for 10 min. The crystal pellet was resuspended in 25 mM Tris-HCl, pH 8.0, and the crystals were washed thoroughly by bringing the crystal suspension to its original volume. The crystals were collected by centrifugation in the same manner. The washing and collecting procedures for rubisco crystals were repeated once. The crystal pellet was completely dissolved in the same buffer containing 0.1 M NaCl. The protein solution was then dialyzed in 25 mM Tris-HCl, pH 8.0 to obtain crystalline rubisco. Crystallization of the New Crystalline Form of Rubisco. The crude leaf extract was obtained in the same manner as Journal of Proteome Research 2002, 1, 471-473
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Simple Purification of Rubisco
technical notes
Figure 2. SDS-PAGE of tobacco leaf extracts from both crystallization procedures. Lanes 1-3 are extracts at pH 5.6, F2P, and rubisco obtained from the new purification procedure. Lane 4, extract in Tris buffer at pH 8.0 after Sepahdex chromatography. Lane 5, F2P after crystallization and crystalline rubisco was removed. Lane 6, rubisco. LS: large subunit of rubisco. SS: small subunit of rubisco. Figure 1. Schematic procedure for simple purification and crystallization of a new crystalline form of rubisco from tobacco leaves.
previously described for the dodecahedral form of rubisco. The pH of the crude leaf extract was adjusted to 5.6 with either 1 N HCl or 1 N NaOH depending upon the initial pH of the extract. The crude extract was centrifuged at 30000g for 30 min. The clarified brown supernatant was then placed in the refrigerator for 24-48 h for crystallization of rubisco. Electrophoresis. SDS-polyacrylamide gel electrophoresis of protein samples was performed in a Bio-Rad (Richmond, CA) Protean II vertical slab gel apparatus according to Chua4 with modifications. Samples containing approximately 50-100 µg protein were loaded onto the gel and were separated in 15% polyacrylamide gel. Gel was stained and destained as previously described.5
Results and Discussion Simple Procedure for Purification and Crystallization of Rubisco. Figure 1 outlines the procedure for purification and crystallization of rubisco from tobacco leaves. Reducing agents, other than sodium meta-bisulfite, such as β-mercaptoethanol and dithiothreitol can also be used to obtain the same result.2,3 In this experiment, however, Na2S2O5 was used as it is generally recognized as safe (GRAS) for food application. In addition, the low cost of Na2S2O5 will be important in planning for future large-scale preparation of crystalline rubisco. For convenience, 200 mL of 2% sodium meta-bisulfite (Na2S2O5) solution was used for homogenizing 400 g of leaf tissue. Heat treatment of crude extract at 40 °C for 10 min prior to centrifugation was reported and previously employed to facilitate the removal of green pigments and their thylakoid membranes.3 However, it has been reported recently that unfolding/aggregation of rubisco activase may occur with heat treatment at 37 °C.6 We also eliminated this heating step because it caused significant reduction in rubisco and total protein in the leaf extract (data 472
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Figure 3. New form of crystalline rubisco, 1260× magnification.
not shown). In addition, heating could potentially be harmful to the L1 protein. Complete Removal of Rubisco from the Leaf Extract. After the supernatant was stored for 48 h at 4 °C, crystalline rubisco had formed and mostly settled to the bottom of container. Rubisco was separated from other soluble protein in the supernatant that is generally known as fraction-2 protein (F2P). Separation was achieved by centrifugation at 3000g for 10 min. As demonstrated by SDS-PAGE in Figure 2, rubisco is removed from F2P by this simple purification procedure. In contrast, a large amount of rubisco remains in F2P when the Sephadex chromatography technique is employed to obtain the dodecahedral form of rubisco. Conversion of New Rubisco Crystals to Its Common Dodecahedral Form. Figure 3 shows the rubisco crystals obtained by this simple technique are not well-ordered. The crystals lack well-defined facets or edges as compared to the original dodecahedral form obtained through Sephadex chromatography technique. However, the crystals were uniform in
technical notes
Kwanyuen et al.
In conclusion, this simple procedure has enabled the purification and crystallization of rubisco without desalting and/ or removing polyphenols using Sephadex chromatography or the generation of large volumes of eluate. Additionally, the use of Tris-HCl buffer and other chemicals that was previously reported3 for obtaining the dodecahedral form of rubisco has been eliminated by the new procedure. Avoiding the use of chemicals for the purification of rubisco is beneficial to the future large-scale production of rubisco for medicinal purposes and/or food applications. The new crystallization procedure has resulted in complete removal of the abundant quantity of rubisco found in the leaf protein extract. Because the presence of rubisco interferes with the purification of the L1 protein, this success has resolved a major problem in the use of transgenic tobacco as a means to bioproduce vaccines. Figure 4. Dodecahedral form of rubisco that has been converted from the new form shown in Figure 3, 400× magnification.
size and shape. This crystal form appeared oval in shape when observed from its side, but square when viewed from the top. To convert this new crystalline rubisco to its common dodecahedral form, the crystals were washed twice in the same manner as described in the Experimental Section for recystallization procedure of the dodecahedral form, but with 25 mM sodium acetate buffer, pH 5.6 instead of Tris-HCl buffer. The crystals pelleted by centrifugation were solubilized in 25 mM Tris-HCl, pH 8.0 containing 0.1 M NaCl. The protein solution was then dialyzed against a large volume of 25 mM Tris-HCl, pH 8.0 to remove NaCl. In our experiment, the ratio of dialysis buffer to protein solution was 20:1 (v/v). We observed that the dodecahedral crystals started to form from this protein solution within 5 min, and crystallization was completed within 1 h. Microscopic examination of these crystals indicated that they all are in dodecahedral form as shown in Figure 4. Conversion and recrystallization can also be performed using Milli-Q purified water, and the same result is obtained.
Acknowledgment. This work was a cooperative research effort between the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service. We are grateful to Dr. Nina Allen and her staff (Mr. Jeff Xu) for the photomicrography of the crystals. References (1) Hiatt, A.; Cafferkey, R.; Bowdish, K. Production of antibodies in transgenic plants. Nature 1989, 342, 76-78. (2) Chan, P. H.; Sakano, K.; Singh, S.; Wildman, S. G. Crystalline Fraction I Protein: Preparation in Large Yield. Science 1972, 176, 1145-1146. (3) Lowe, R. H. Crystallization of Fraction I Protein from Tobacco by a Simplified Procedure. FEBS Lett. 1977, 78, 98-100. (4) Chua, N.-H. Electrophoretic Analysis of Chloroplast Proteins. Methods Enzymol. 1980, 69, 434-446. (5) Kwanyuen, P.; Wilson, R. F. Optimization of Coomassie Staining for Quantitative Densitometry of Soybean Storage Proteins in Gradient Gel Electrophoresis. J. Am. Oil Chem. Soc. 2000, 77, 1251-1254. (6) Salvucci, M. E.; Osteryoung, K. W.; Crafts-Brandner, S. J.; Vierling, E. Exceptional Sensitivity of Rubisco Activase to Thermal Denaturation in Vitro and in Vivo. Pl. Physiol. 2001, 127, 1053-1064.
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