Computer Software Virtual Protein Purification: A Simple Exercise to Introduce ph as a Parameter that Effects Ion Exchange Chromatography ws

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1 Computer Software Virtual Protein Purification: A Simple Exercise to Introduce ph as a Parameter that Effects Ion Exchange Chromatography ws Daniel D. Clark * Daniel J. Edwards From the Department of Chemistry and Biochemistry, California State University-Chico, Chico, California Abstract This article describes a simple exercise using a free, easy-touse, established online program. The exercise helps to reinforce protein purification concepts and introduces undergraduates to ph as a parameter that affects anion-exchange chromatography. The exercise was tested with biochemistry majors at California State University-Chico. Given the versatility of the program, this work is also a model for instructors that wish to develop their own exercise to help teach other protein purification techniques. VC 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):91 97, Keywords: Protein purification; ion exchange; computer software; web-assisted learning Introduction Protein purification is an important topic in the undergraduate biochemistry curriculum. In a lecture setting, a discussion of ion exchange, size-exclusion, and affinity-based purification techniques is common. In the laboratory, the implementation of one or more of these techniques is also common. However, despite this coverage, undergraduates typically do not have an opportunity to explore key chromatographic parameters, for a particular media (or resin) type, that can affect a protein separation. To afford students this kind of experience, not available from one iteration of a supplied protocol, would enhance their conceptual understanding and practical ability to execute a protein Volume 46, Number 1, January/February 2018, Pages Abbreviations: FPLC, fast protein liquid chromatography; DEAE-cellulose, diethylaminoethyl-cellulose; CM-cellulose, carboxymethyl-cellulose; Q-Sepharose, trademarked (GE Healthcare) resin; Q refers to quaternary amine; S-Sepharose, SP-Sepharose, trademarked (GE Healthcare) resin; S refers to sulfopropyl; LC-MS/MS, liquid chromatography-tandem mass spectrometry *To whom correspondence should be addressed. Department of Chemistry and Biochemistry, California State University-Chico, 400 West First Street, Chico, CA Tel.: (530) ddclark@csuchico.edu ws Additional Supporting Information may be found in the online version of this article. Received 10 April 2017; Revised 13 June 2017; Accepted 18 July 2017 DOI /bmb Published online 6 August 2017 in Wiley Online Library (wileyonlinelibrary.com) purification. While actual lab time may never permit it, a virtual lab experience can easily facilitate the exploration of chromatographic parameters in a rapid manner. Herein we present a simple virtual protein purification exercise that utilizes a free, established online program [1]. The primary objectives of the exercise were to (1) reinforce previously learned protein purification concepts (specifically ion exchange), and (2) introduce students to ph as a parameter that effects anion-exchange chromatography. A secondary objective was to have students discover the value of investigating both strong (Q) and weak (DEAE) anionexchange media as part of a protein purification. We sought to accomplish these objectives through six protein purification simulations that would generate realistic FPLC chromatograms for students to think about, discuss, and interpret. Background Proteins are purified using techniques that separate them according to differences in charge, size, hydrophobicity, and molecular recognition (affinity). Ion exchange chromatography separates proteins based on differences in net surface charge, which is highly ph dependent [2]. The isoelectric point (pi) of a protein is the ph at which it carries no net charge. Protein titration curves indicate that at ph values less than the pi, a protein will carry an increasing net positive charge, while at ph values greater than the pi, a protein will carry an increasing net negative charge [2]. Ion exchange chromatography includes anion and cation exchangers. Anion exchangers employ a positively charged resin interacting with an exchangeable anion, while cation Biochemistry and Molecular Biology Education 91

2 Biochemistry and Molecular Biology Education exchangers employ a negatively charged resin interacting with an exchangeable cation. A strong anion exchanger, such as Q-Sepharose is positively charged across a broad ph range, whereas a weak anion-exchanger, such as DEAEcellulose will lose its charge above ph 9 [3]. Therefore, column equilibration ph is an important parameter in ion exchange chromatography as it can affect protein charge and, depending on the resin, the charge on the column. Both of these affect protein adsorption and separation. Program Description Protein Purification is a free online program developed by the late (June 2016) Dr. Andrew G. Booth at the University of Leeds ( [1]. The program offers four protein mixtures of varied complexity to explore: Easy3 (three proteins), Example (six proteins), Default (20 proteins), and Complex (60 proteins). Users can select any of the proteins within a mixture to purify. The separation techniques available are ammonium sulfate fractionation, heat treatment, gel filtration, ion exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. Within each of these techniques, users can alter various parameters. In the case of ion exchange chromatography, anion-exchange media (Q- Sepharose and DEAE-cellulose) or cation-exchange media (S-Sepharose and CM-cellulose) are available. With either, users define the method of elution as either a salt gradient or ph gradient. With salt gradient selected, users can define the ph of the equilibration buffer ( ) and the start and end salt concentrations within a 0 to 3.0 M range. When a column-based purification technique is chosen, a FPLC chromatogram appears that displays absorbance at 280 nm (A 280 ) on the primary y-axis, fraction number on the x-axis, and [salt] on the secondary y-axis (see Fig. 1). Although not employed in our exercise, the program also features virtual 1D- and 2D-gels (with coomassie blue and immunoblot staining available) that enable users to analyze fractions from a column. The program also contains a help feature that includes six tutorial exercises, but practice with these is not required for the exercise described in this work. A literature search indicates that two publications using the program are available, and both are from the author of the program [1, 4]. More recently, a web-based applet that simulates an ion exchange purification of over produced proteins from E.coli was described [5]. However, we decided to use the program devised by Dr. Andrew Booth for its ease of use and versatility that extends to techniques beyond ion exchange chromatography [1, 4]. Exercise Description In an effort to keep the exercise simple, we designed it with a focus on anion-exchange chromatography. It can be completed without instructor help in about an hour. The exercise as provided to students (see Supporting Information, pages 2 4) includes Part 1, a concept review, and Part 2, the virtual ion exchange chromatography, which uses the online program. In Part 2 students use the threecomponent Easy3 protein mixture to execute six virtual ion exchange separations. The simulations (A F) vary the ion exchange media, equilibration ph, and the salt gradient as follows: (A) Q-Sepharose, ph 6.0, M salt, (B) Q- Sepharose, ph 7.0, M salt, (C) Q-Sepharose, ph 8.0, M salt, (D) Q-Sepharose, ph 10.0, M salt, (E) Q- Sepharose, ph 10.0, M salt, (F) DEAE-Cellulose, ph 10.0, M salt. In each simulation, students use the assay enzyme activity feature to locate fractions that contain Protein 1. Students draw the results as a FPLC chromatogram on the appropriate panel (A F) of a six-panel figure in the exercise (see Supporting Information, page 3). Their chromatograms include an A 280 trace, the linear salt gradient, and the location of Protein 1. Additionally students use the linear salt gradient to estimate the [salt] (within 25 m M) required for elution of Protein 1. Figure 1 shows the six screen shots (A F) that students would generate using the program and the annotations (*) that students would use to indicate the location of Protein 1, and the estimated [salt] required to elute Protein 1. Table 1 presents the seven questions that follow use of the program, our rationale for their inclusion, and an evaluation of student performance on each question. Implementation At California State University-Chico, we tested this exercise in our seven hours per week, biochemistry lab (designed for biochemistry majors) during the Spring 2017 semester. All of the students had previously learned about protein purification, including ion exchange chromatography, in lecture. In the schedule of the course, the exercise preceded two major projects (1) a scripted three-week purification and characterization of a recombinant fluorescent protein, and (2) an unscripted seven-week student designed project aimed at the purification, identification (via LC-MS/ MS), and characterization of a dehydrogenase enzyme from its native source. The exercise was presented during a three hour lab as the prelude to an open-ended dry lab protein purification experience (described below) using the same program. Each student completed the exercise individually without instructor help. Immediately afterwards, each student s attitudes toward the exercise were assessed by way of an anonymous survey (Table 2). Following assessment, each student was assigned a protein (1 60) at random from the complex mixture and had about two hours to optimize a purification strategy. Importantly, at this stage, students could explore the variety of techniques the program offers, use virtual gels, and pool fractions. To increase the difficulty, and emphasize the 92 Virtual Protein Purification

3 FIG 1 Screen shots of FPLC chromatograms using the program. Each simulation used the Easy3 mixture. Asterisks indicate the location of Protein 1 with the estimated [salt] required for elution given. Ion-exchange media, equilibration ph, and the salt gradient varied as follows:(a) Q-Sepharose, ph 6.0, M salt, (B) Q-Sepharose, ph 7.0, M salt, (C) Q- Sepharose, ph 8.0, M salt, (D) Q-Sepharose, ph 10.0, M salt, (E) Q-Sepharose, ph 10.0, M salt, (F) DEAE- Cellulose, ph 10.0, M salt. Panels lettered according to the exercise (see Supporting Information, page 3). [Color figure can be viewed at wileyonlinelibrary.com] other available techniques, affinity chromatography was not allowed. As analytical tools to guide their purifications, only the assay enzyme activity and 1D-gel (coomassie blue) features were permitted. The greater purpose for these restrictions was to align the experience with their seven-week student designed purification later in the semester. Optimization focused on employing a logical sequence of purification steps (for example, size-exclusion Clark and Edwards 93

4 Biochemistry and Molecular Biology Education TABLE 1 Exercise questions, rationale for inclusion, and student performance on each question Questions Rationale for inclusion Percentage of effective student explanations (%) a 1. Examine the chromatograms for Q-Sepharose run at ph 6 and 7 (Panels A & B). Where did Protein 1 elute in both cases? Explain the basis for the observation. 2. Examine the chromatograms for Q-Sepharose run at ph 6, 7, and 8 (Panels A C). What happened to the [salt] required for the elution of Protein 1 as the equilibration ph increased? Explain the basis for the observation. 3. Examine the chromatogram for Q-Sepharose run at ph 10, specifically the one that employed a M NaCl gradient (Panel D). At what [salt] did Protein 1 elute? Explain the basis for the observation. 4. Examine the chromatogram for Q-Sepharose run at ph 10, specifically the one that employed a 0 1 M NaCl gradient (Panel E). At what [salt] did Protein 1 elute? How does this result compare to the M NaCl gradient (Panel D)? Explain the basis for any observed differences. 5. Compare the chromatograms for Q-Sepharose (Panel D) and DEAE-cellulose (Panel F) run at ph 10. How do they compare? Explain the basis for any observed differences. 6. Examine the chromatogram for DEAE-cellulose run a ph of 10 (Panel F). How might the results have changed if the equilibration ph was lowered to ph 8? Try it with the program and explain the basis for the observed result. 7. Use the chromatograms to estimate the pi of Protein 1. Explain how you did this. Students are required to think about two situations where Protein 1 adsorption did not occur. They will need to consider the charge on the resin and the charge on Protein 1 at each ph value. Students discover that an increase in the equilibration ph led to a stronger Protein 1-column interaction and this necessitated a higher [salt] for elution. Their explanation will require consideration of how a higher ph might have strengthened the interaction. Students are required to consider a situation where Protein 1 elution did not occur and think about why this happened. Students discover that a strong Protein 1-column interaction can be overcome by increasing the final [salt] of the linear gradient. The comparison illustrates differences between strong (Q-Sepharose) and weak (DEAE-cellulose) anion exchangers. An effective answer will require students to consider the structure of the ion exchange groups on each resin. Students will discover that Protein 1 does not adsorb to DEAE-cellulose at ph 10, but does at ph 8. An explanation will require students to think about the structure of the DEAE-cellulose ion exchange group, how its charge is affected by ph, and how this relates to Protein 1 adsorption. This question requires student reflection on all of the chromatograms, which further reinforces ph as a parameter that effects anion-exchange chromatography a Percentages calculated using the student exercises that were available for analysis (n 5 6). 94 Virtual Protein Purification

5 TABLE 2 Postexercise assessment of student attitudes Student response percentages (%) a Question SA A N D SD 1. This exercise increased my understanding of ion exchange chromatography as part of a protein purification. 2. This exercise increased my understanding of how equilibration ph can affect protein binding to and elution from, an anion-exchange column. 3. This exercise helped me to understand how protein binding and elution can vary between Q and DEAE anion-exchange columns. 4. Although this exercise focused on anion-exchange chromatography, I feel it gave me the foundation to understand how equilibration ph can affect protein binding to and elution from, a cation-exchange column. 5. This exercise should be part of future iterations of the course a Strongly agree (SA), agree (A), neither agree nor disagree (N), disagree (D), strongly disagree (SD). Percentages calculated using data from all students in the course (n 5 8). is not an appropriate first step) and a reduction in the total number of steps, but was not concerned with yield or cost (in person-hours), which the program tracks. Students were required to illustrate their optimized strategy with a detailed flow diagram that included (if applicable) the separation technique, type of chromatography resin, equilibration ph and type of gradient used, which fractions were pooled and the salt concentration range that the pool encompassed. Additionally, students completed a purification table and determined the pi and molecular weight of their purified protein using a 2-D gel. In fact, a 2D-gel (coomassie blue or immunoblot) was only authorized on the purified protein obtained via their optimized strategy for the express purpose of pi and molecular weight determination. Therefore, as it would be for a new protein (whose sequence was unknown), pi information was not directly available to guide the development of their purification. Evaluation of Student Performance The evaluation of student performance on each of the exercise questions is provided in Table 1. Provided as Supporting Information (Table S1, pages 5 6), are examples of high quality and low quality student answers, to each of the seven exercise questions in Table 1. Effective student explanations, indicated by percentage in Table 1, were those that were well articulated and on trajectory with the high quality answers provided in Supporting Information Table S1. As shown in Table 1, at least 50% of the available student explanations were effective for any given question. These percentages would likely have been higher if instructor student and student student interactions were permitted during the exercise. While such interactions are synergistic and would normally be desirable, for the purpose of this work, we chose to forbid them to control influences on student performance data (Table 1) and student attitude data (Table 2). It is noteworthy that 100% of the available student explanations were effective for Question 7, as it required reflection on all of their chromatograms to estimate the pi (Table 1). As it was beyond the focus of the present work, the purification of a protein from the sixty component complex mix was not targeted for assessment. Despite this, we wish to offer some insight into how students performed. All students successfully completed their optimized purification in under two hours with a couple of students finishing in about an hour. The task challenged students, but by deemphasizing yield and cost, it was readily achievable. Each student performed many iterations before arriving at an optimized protocol. The techniques that students often employed were ammonium sulfate fractionation, heat Clark and Edwards 95

6 Biochemistry and Molecular Biology Education TABLE 3 Postexercise assessment of student learning Question: A certain protein has a pi of 7.5. At a ph of 7.0 this protein would be expected to: Percentage of students selecting an answer (%) a (a) Adsorb to an anion exchange column 25 (b) Adsorb to a cation exchange column (CORRECT RESPONSE) 75 (c) Adsorb to a size exclusion column 0 (d) Adsorb to a hydrophobic interaction column 0 (e) Cannot be predicted without more information 0 a Percentages calculated using data from all students in the course (n 5 8). treatment (only for those with proteins stable at 508C or greater), hydrophobic interaction, and size-exclusion. In terms of basic strategy, ion exchange, followed by hydrophobic interaction, then gel filtration, proved successful in many cases. As mentioned previously, students were not allowed to use a 2D-gel to find the pi of their target protein, which would have helped guide their purifications. Interestingly, a few students estimated pi indirectly, as they did with the exercise (see Table 1, Question 7), by examining their target protein s ph-dependent behavior on an ion exchange column. These students subsequently used this information to assist the design of specific ion exchange steps. Given that many aspects of this open-ended purification were new to students, instructor guidance and interaction appeared to be essential for success. Yet, such guidance was expected, and it provided an opportunity for students to learn in lieu of their wet-lab purifications later in the semester. equilibration ph can affect protein binding to and elution from, a cation-exchange column (Table 2). The postexercise assessment of student learning is shown in Table 3. The question was part of a comprehensive final examination given three and a half months after students completed the exercise. The results indicated that the majority of students (75%) correctly predicted that a protein with a pi of 7.5 would adsorb to a cation exchange column at ph 7.0 (Table 3). To answer this question, students would have had to consider the net charge on the protein at this ph, and the charge on the chromatography column both of which were fundamental concepts emphasized in the exercise. This result aligned with the postexercise assessment of student attitude, where the majority of students (87.5%) felt that the exercise gave them the foundation to understand how equilibration ph can affect protein binding to and elution from, a cation-exchange column (see Table 2, Question 4). Assessment The postexercise assessment of student attitudes via an anonymous survey, which employed a five-point Likert scale, is shown in Table 2. The sample size (n 5 8) reflected the small number of students that take this course at CSU Chico; for example, total enrollment in Fall 2016 and Spring 2017 semesters were seven and eight students, respectively. Student responses reflected a favorable view of the exercise (Table 2). Question 4 was included to gauge student confidence with what they had learned and whether they thought they could extend their knowledge to cation-exchange chromatography (Table 2). The data for Question 4 suggested that most students felt the exercise had given them the foundation to understand how Summary This article describes a unique exercise using a free online program. The exercise focuses on anion-exchange chromatography and utilizes a simple protein mixture. However, the versatility of the program affords the creation of similar exercises with other chromatographic techniques and more complex protein samples. Therefore, our work also serves as a model for instructors to develop their own custom exercise to augment their teaching of protein purification. Such an exercise can be implemented in a lecture course, as a pre-lab assignment, or as an in-lab prelude to a wetlab or dry-lab (as we have done). We do not advocate for a virtual experience to replace an actual protein purification. Instead, we believe this program significantly enhances any 96 Virtual Protein Purification

7 wet-lab experience by giving students the opportunity to explore chromatographic parameters to a degree not possible within the time constraints of a normal lab. References [1] Booth, A. G. (1986) Simulation of protein separation techniques on a personal computer. Biochem. Soc. Trans. 14, [2] GE Healthcare Life Sciences. Handbook: Ion Exchange Chromatography Principles and Methods. servlet/catalog/en/gelifesciences-us/service-and-support/handbooks/ (accessed March 6, 2017). [3] Scopes, R. K. (1994) Protein Purification: Principles and Practice, 3rd ed., Springer-Verlag, New York. [4] Phornphisutthimas, S. Panijpan, B. Wood, E. J., and Booth, A. G. (2007) Improving Thai students understanding of concepts in protein purification by using Thai and English versions of a simulation program. Biochem. Mol. Biol. Educ. 35, [5] Usher, K. C., and Barrette-Ng, I. H. (2012) Web-based applet is a learning tool that simulates ion exchange chromatography purification of overexpressed proteins from Escherichia coli cell lysate. J. Chem. Educ. 89, Clark and Edwards 97