Technology Report pubs.acs.org/jchemeduc
Web-Based Applet Is a Learning Tool that Simulates Ion-Exchange Chromatography Purification of Overexpressed Proteins from Escherichia coli Cell Lysate Ken C. Usher*,† and Isabelle H. Barrette-Ng‡ †
Department of Natural Sciences, Oregon Institute of Technology, Klamath Falls, Oregon, 97601, United States Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4
‡
S Supporting Information *
ABSTRACT: A Java Web applet is described that helps biochemistry students learn how varying ionic strength and pH can affect separation of proteins in an ion-exchange chromatography experiment. The applet simulates a common scenario: a foreign protein is overexpressed in bacteria and needs to be isolated from the other water-soluble proteins of the cell lysate. It allows visualization of proteins on the column, a protein output graph (absorbance at 280 nm), and polyacrylamide gel electrophoresis analysis of selected output fractions. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Internet/Web-Based Learning, Chromatography, Electrophoresis, Ion Exchange, Proteins/Peptides
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Java1 Web applet is described2 that helps biochemistry students learn how varying ionic strength and pH can affect separation of proteins in an ion-exchange chromatography experiment. The applet simulates a common scenario: a foreign protein is overexpressed in bacteria and needs to be isolated from the other water-soluble proteins of the cell lysate. It allows visualization of proteins on the column, a protein output graph (absorbance at 280 nm), and polyacrylamide gel electrophoresis (SDS−PAGE) analysis of selected output fractions (Figure 1). Biochemistry laboratory courses often include protein purification via chromatography, but the experiment and analysis usually take two weeks, one for the chromatography and another to analyze fractions via SDS−PAGE.3 Often, students follow a prescribed procedure, with little or no opportunity for them to explore on their own. This applet is a useful accompaniment to such laboratories, because it allows students to quickly test how changes in pH and salt concentration gradients affect the separation of a complex mixture of proteins away from a specific target protein.4 The applet is unique among lab simulation software: the most comparable protein purification simulation5,6 is less detailed about ion exchange and less visually oriented. The applet’s cell lysate mixture contains the fifty most abundant water-soluble, cytoplasmic, nonribosomal proteins from Escherichia coli, in experimentally determined proportions,7 and the target protein can be selected from over twenty possibilities. The mobility of each protein is determined by its net charge at the selected pH and by the salt concentration. Accurate protein mobility simulation requires detailed modeling based on three-dimensional structure data,8 but this applet achieves a plausible approximation based on amino acid sequence and previously described models.9,10 Students can use the Java applet to simulate the separation of the protein mixture using the default parameters, or they can © 2012 American Chemical Society and Division of Chemical Education, Inc.
change the salt gradient, the pH, and the use of anion or cation exchange. Changing any of these parameters affects how tightly the target protein binds to the column and the effectiveness of the chromatographic separation. The visual display of the column shows things happening inside that are usually invisible in an actual experiment: the protein bands are visible moving down the column and the column changes color to indicate the increasing salt concentration flowing through it as the simulation proceeds. After running the column, students pick fractions by clicking on peaks in the protein output graph, then click the “Run SDS−PAGE” button to analyze whether those fractions contain one pure protein or a mixture of several proteins. Students can instantly reset the simulation to change parameters and try the experiment again.
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ASSOCIATED CONTENT
S Supporting Information *
The Java applet; overview; prelab instructions; prelab assignment; salt gradients; description of the buttons. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
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REFERENCES
(1) Sun Microsystems - Java Runtime Environment . http://www. java.com/ (accessed Dec 2011). (2) Web-Based Applet . http://media.oit.edu/ken.usher/chrom.html (accessed Dec 2011). (3) Miller, S; Indivero, V; Burkhard, V. Expression and Purification of Sperm Whale Myoglobin. J. Chem. Educ. 2010, 87 (3), 303−305, DOI: 10.1021/ed8000842.
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Technology Report
Figure 1. Screen shot of the Chrom applet. (left) Information about the currently selected target protein and the control buttons for the simulation. (center) Graph showing the chosen salt gradient in blue and the protein output from the column in red, with the target protein output in green. (center-right) Ion-exchange column simulation and a list of which fractions were selected for SDS−PAGE. (right) SDS−PAGE simulation, with molecular weight markers shown in blue. (4) Usher, K. C.; Barrette-Ng, I. H. J. Chem. Educ. 2012, submitted for publication. (5) Booth, A. G. Simulation of protein purification techniques on a personal computer. Biochem. Soc. Trans. 1986, 14, 908−909, DOI: 10.1042/bst0140908a. (6) Protein Purification . http://home.btconnect.com/agbooth/ archive/ (accessed Dec 2011). (7) Ishihama, Y.; Schmidt, T.; Rappsilber, J.; Mann, M.; Hartl, F. U.; Kerner, M. J.; Frishman, D. Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics 2008, 9, 102 DOI: 10.1186/ 1471-2164-9-102. (8) Ladiwala, A.; Rege, K.; Breneman, C. M.; Cramer, S. M. A priori prediction of adsorption isotherm parameters and chromatographic behavior in ion-exchange systems. Proc. Natl. Acad. Sci. U.S.A 2005, 102 (33), 11710−5, DOI: 10.1073/pnas.0408769102. (9) Yamamoto, S; Nakanishi, K; Matsuno, R; Kamikubo, T. Ion exchange chromatography of proteins-prediction of elution curves and operating conditions. I. Theoretical considerations. Biotechnol. Bioeng. 1983, 25 (6), 1465−83. (10) Yamamoto, S; Nakanishi, K; Matsuno, R; Kamijubo, T. Ion exchange chromatography of proteins-predictions of elution curves and operating conditions. II. Experimental verification. Biotechnol. Bioeng. 1983, 25 (5), 1373−91.
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