In the Laboratory
Preparation, Purification, and Secondary Structure Determination of Bacillus circulans Xylanase
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A Modular Laboratory Incorporating Aspects of Molecular Biology, Biochemistry, and Biophysical Chemistry Sal Russo and Lisa Gentile*,† Department of Chemistry, Western Washington University, Bellingham, WA 98225-9150; *
[email protected] Students studying biochemistry or cellular and molecular biology at this university are required to take two quarters of upper-division labs in biochemistry and molecular biology in either their third or fourth year. Characterization of proteins is the basis of the biochemistry lab and is the focus of the module described here. During the quarter, the biochemistry lab meets for two, four-hour blocks as well as an additional hour of lab lecture each week for a total of nine weeks. The experiments are done in pairs in each section of sixteen students. The motivation behind the development of this six-week lab is severalfold. The first was to move away from labs focused on single techniques in the context of different proteins, to an integrated lab with a single protein that allows students to learn techniques that culminate in a discoveryoriented project. Aspects of this basic approach have been documented in this Journal (1–12). The module described here is unique primarily in the protein studied as well as in the focus of the student-designed project. The discovery-oriented portion of this project involves determining the secondary structure of Bacillus circulans xylanase (BCX) by circular dichroism (CD) spectroscopy under conditions that compromise its stabilizing intramolecular forces. CD spectroscopy is a valuable technique for monitoring changes in protein secondary structure, but it is not often incorporated into undergraduate biochemistry labs (13, 14). Incorporation here also supports the curriculum for the biochemistry majors by providing them with hands-on experience using a CD spectrometer. The theoretical and practical aspects of CD analysis are also discussed in a required biophysical chemistry lecture course that is not a prerequisite to this lab course. Other benefits of this lab are that students gain experience in scientific writing and in understanding some of the tools available to biochemists on the Internet. For each week of the module, a prelab is due that is written in the format of an article in Biochemistry. It includes an abstract, introduction, and a materials and methods section. In the introduction section, students must explain specific topics after obtaining background information on them. The frequent submission of prelabs ensures that students get the help needed writing scientific reports. The final lab report, also written in the same format, includes all sections of each prelab as well as the results and discussion section. Finally, incorporation of aspects of molecular biology, biochemistry, and †
biophysical chemistry into one lab module allows students to see the relationship between disciplines. BCX was chosen for this series of experiments primarily because it is highly over-expressed (∼75 mg of pure protein from 1 L of growth), easily purified in a single chromatographic step, and stable (fully folded in 7.5 M urea). It is a member of the family 11 or G, low molecular weight endoβ-(1,4)-xylanases, which preferentially hydrolyze xylose polymers yielding xylobiose and xylotriose (15). Xylanolytic enzymes have many applications, including clarification of juices and wines, saccharification of agricultural and forestry wastes for fermentation to fuels, improvement of the nutritional value of agricultural silages, and conversion of xylans to simple xylose derivatives used, for example, as sweeteners. Accordingly, the focus of xylanase research is aimed both at understanding the mechanisms by which they bind and catalyze hydrolysis of xylan and at exploiting these enzymes for their effective use in biotechnology (15–17). Course Schedule
Lab Periods 1–3: Preparation of Competent Cells and Transformation Table 1 outlines the instrumentation, techniques, and lab lecture topics for each lab period of this module. Before beginning, students need to learn how to make media, pour plates, and the essentials of sterile technique. Once mastered, chemically competent E. coli 594 cells are prepared and transformed with the plasmid pCW containing the structural gene for BCX (16, 17). Transformations are plated out on LB-Amp plates (the plasmid pCW encodes for ampicillin resistance) to select for cells containing the desired plasmid. Lab Periods 4–6: Protein Over-Expression and Purification A single colony of E. coli 594 cells transformed with pCW兾BCX is then grown for two purposes: protein overexpression and plasmid purification. Isopropylthio-β-D-galactoside (IPTG) is used to induce protein expression from the tac promoter in pCW, and over-expressed BCX is harvested. The E. coli cells are lysed by sonication, and the high speed supernatant and pellet are separated by centrifugation. The protein is applied to an SP-sepharose cation-exchange column (calculated pI of BCX: 9.1) and eluted with 50 mM NaCl at pH 6.0 (16, 17).
Current address: Department of Chemistry, University of Richmond, Richmond, VA 23173.
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In the Laboratory
Table 1. Instrumentation, Techniques, and Lab Lecture Topics Lab Period, Experiment
Instrumentation
Techniques
Lab Lecture Topic(s)
1–3. Preparation of competent E. coli cells and transformation
Spectronic-20, high speed centrifuge
Sterile technique, bacterial growth, preparation of competent cells, DNA transformation, negative controls
Cloning overview, including: PCR, plasmids, regulation of protein over-expression
4–6. Protein over-expression and purification by ion exchange chromatography
High speed centrifuge, protein chromatography system, UV–vis spectrophotometer
Plasmid preparation, protein induction, sonication, ion exchange chromatography
Chromatography
7–8. Electrophoresis and online studies
Protein electrophoresis apparatus
SDS-PAGE, protein quantitation, structure prediction tools, protein alignment tools, Internet biochemical calculations, literature searching
Electrophoresis, protein quantitation
9–12. CD- based independent projects
Circular dichroism spectrometer
Determination of protein secondary structure
CD and forces holding proteins together
Lab Periods 7–8: Protein Electrophoresis, Quantitation, and Online Studies The fractions from the cation-exchange column are analyzed for protein content and purity (SDS-PAGE, BCX: 20,395 Da). The activity of BCX can be verified using a fluorescence-based assay (EnzChek Ultra, Invitrogen). Pure active fractions are pooled and quantitated (∼30 mL of 2–3 mg兾mL pure BCX from 1 L of preparation). In addition, online tools for biochemists focusing on BCX (homology searches, pI and molecular weight calculations, secondary structure predictions, analysis of PDB files for experimentally determined secondary and tertiary structures, and literature searches) are explored. The results from these studies (molecular weight calculation and predicted and experimentally determined secondary structure) are then compared to those experimentally determined in this module. Lab Periods 9–12: Independent Projects In the last two weeks, students design a CD-based experiment to probe the stability of BCX. They are encouraged to be as creative and original as possible in designing a hypothesis about how to influence the forces holding BCX together. The native and perturbed secondary structures of BCX are monitored by CD spectroscopy, primarily by examining the β-sheet signal (218 nm) of BCX. Examples of student-designed experiments include denaturation by alcohols of various concentrations and chain lengths, detergents, heavy metals, nonalcoholic organic solvents, chemical denaturants, heat, cross-linking with glutaraldehyde, and sonication. Hazards The hazards for this lab include the QIA miniprep kit: sodium hydroxide is corrosive to all tissues; guanidine hydrochloride causes eye and skin irritation; glacial acetic acid is corrosive and causes severe burns; and isopropanol is an eye irritant. Acrylamide is a suspected human carcinogen and severe neurotoxin and causes irritation of the eyes, skin, and respiratory tract. Sodium dodecyl sulfate causes eye and skin irritation. Ammonium persulfate may cause sensitization by
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inhalation and skin contact. N,N,N´,N´-tetramethylethylenediamine is corrosive, causes burns, and is harmful by inhalation. β-Mercaptoethanol causes eye and skin irritation. Methanol is toxic by inhalation and if swallowed. Future Plans As the university instrument holdings increase, we envision incorporating additional modules into this lab. For example, the recent acquisition of an ESI mass spectrometer and a fluorimeter will allow these units to be incorporated next year. Note, as written, this module requires a CD spectrometer. For departments without a CD spectrometer, the final two weeks of the lab could focus on available instrumentation. For example, protein secondary structure can be monitored by FT-IR or tertiary structure by fluorescence spectroscopy. Alternatively, a unit focusing on enzyme kinetics could be incorporated. Results and Discussion Students have been successful in preparing competent E. coli 594 cells, transforming them with the plasmid pCW兾BCX, over-expressing BCX, and purifying the protein by cation-exchange chromatography. The results of two of the many interesting projects are shown in Figure 1. Figure 1A shows the results of varying methanol concentration on the secondary structure of BCX. When BCX is in 75% (v兾v) methanol, there is ∼30% more β-sheet secondary structure present (determined by CD signal at 218 nm) compared to native BCX. In 80% methanol the secondary structure of BCX is virtually identical to that of native BCX, and in 90% methanol there is significant denaturation. Figure 1B shows that heat is a more effective denaturant of BCX than is urea. In 7.5 M urea, the secondary structure of BCX is native-like, while at 100 ⬚C, BCX is denatured. Results such as those described above are interesting in that they can lead to a wide range of discussions on the primarily noncovalent forces that hold proteins together and how they might be disturbed or altered upon addition of various reagents. In addition to enhancing their knowledge of the theoretical aspects of protein stability, students gain ex-
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In the Laboratory A
B
Figure 1. Far-UV CD spectra of BCX in 10 mM sodium phosphate, 50 mM NaCl, pH 6.0: (A) 6.5 µM BCX: native BCX (䊐); BCX in 75% v/v MeOH (䊉); BCX in 80% v/v MeOH (䊏); BCX in 90% v/v MeOH (䉱) and (B)13 µM BCX: native BCX at room temperature (䊐); BCX in 7.5 M urea at room temperature (䊏); BCX at 100 oC (䉱).
perience with instrumentation and molecular biology techniques needed to over-express protein from a recombinant DNA system, to purify and quantitate protein, and to characterize the secondary structure of proteins. This module aids in student learning for several reasons: (i) by engaging students in multiple techniques in the context of a single protein, they understand how each technique contributes to the overall production and characterization of the protein, (ii) by allowing students to design a CD-based experiment for the last two weeks of the module, they are required to explore more deeply the forces responsible for protein stability, (iii) because students both compare their experimentally determined values to those found in the literature and also use the literature to aid in the design of their CD-based experiment, they gain a better understanding of the data available in the primary literature and how it is determined, and (iv) by combining molecular biology, biochemistry, and biophysical chemistry into a single lab module, students more easily see connections between the disciplines. Finally, other advantages of this lab from the instructor’s view point are that it can be expanded or modified based on university instrument holdings and that the experience is kept fresh owing to the design of different experiments each year by the students. Based on the final lab report submitted by each student as well as their performance on the final exam, it is clear that students are meeting the expected educational outcomes. Student evaluations of the course indicate that they like the lab module (although no one student has taken the biochemistry lab with and without this module) and the continuity it provides. Although indicating it is more work, students like being able to design part of the lab and interpret their results. A number of students go on to do undergraduate research in biochemistry and in addition to the advantages stated above, this module provides them with a good set of tools from which to start. Acknowledgments The authors thank Warren Wakarchuk (NRC Ottawa) for the pCW兾BCX plasmid and Manish Joshi and Lawrence
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McIntosh (UBC) for E. coli 594 cells and helpful hints on the over-expression and purification of BCX. We also thank one of the reviewers for bringing to our attention the EnzChek Ultra kit from Invitrogen. W
Supplemental Material
Instructor notes, experimental procedures, and student handouts are available in this issue of JCE Online. Literature Cited 1. Shafirovich, V.; Singh, C.; Geacintov, N. E. J. Chem. Educ. 2003, 80, 1297–1299. 2. Taylor, A. T. S.; Feller, S. E. J. Chem. Educ. 2002, 79, 1467– 1470. 3. Parra-Belky, K. J. Chem. Educ. 2002, 79, 1348–1350. 4. Stanish, I.; Zabetakis, D.; Singh, A. J. Chem. Educ. 2002, 79, 481–483. 5. Olchowicz, J.; Coles, D. R.; Kain, L. E.; MacDonald, G. J. Chem. Educ. 2002, 79, 369–371. 6. Chow, C. S.; Somne, S. J. Chem. Educ. 1999, 76, 648–650. 7. Chow, C. S.; Somne, S.; Llano–Sotelo, B. J. Chem. Educ. 1999, 76, 651–652. 8. Hicks, B. W. J. Chem. Educ. 1999, 76, 409–415. 9. Wolfson, A. J.; Hall, M. L.; Branham, T. R. J. Chem. Educ. 1996, 73, 1026–1029. 10. Craig, P. A. J. Chem. Educ. 1999, 76, 1130–1135. 11. Brockman, M.; Ordman, A. B.; Campbell, A. M. J. Chem. Educ. 1996, 73, 542–543. 12. Bonser, A. M.; Moe, O. A. J. Chem. Educ. 1996, 73, 794– 796. 13. Cavaleiro, A. M. V.; Pedrosa de Jesus, J. D. J. Chem. Educ. 2000, 77, 1218–1220. 14. Bondesen, B. A.; Schuh, M. D. J. Chem. Educ. 2001, 78, 1244–1247. 15. Gilkes, N. R.; Henrissat, B.; Kilburn, D. G.; Miller, R. C., Jr.; Warren, R. A. J. Microbiol. Rev. 1991, 55, 303–315. 16. Wakarchuk, W. W.; Campbell, R. T.; Sung, W. L.; Davoodi, J.; Yaguchi, M. Protein Science 1994, 3, 467–475. 17. Sung, W. L.; Luk, C. K.; Zahab, D. M.; Wakarchuk, W. Protein Express. Purif. 1993, 4, 200–216.
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