Using an FPLC to promote active learning of the principles of protein structure and purification
Abstract
The concepts of protein purification are often taught in undergraduate biology and biochemistry lectures and reinforced during laboratory exercises; however, very few reported activities allow students to directly gain experience using modern protein purification instruments, such as Fast Protein Liquid Chromatography (FPLC). This laboratory exercise uses size exclusion chromatography (SEC) and ion exchange (IEX) chromatography to separate a mixture of four different proteins. Students use an SEC chromatogram and corresponding SDS-PAGE gel to understand how protein conformations change under different conditions (i.e. native and non-native). Students explore strategies to separate co-eluting proteins by IEX chromatography. Using either cation or anion exchange, one protein is bound to the column while the other is collected in the flow-through. In this exercise, undergraduate students gain hands-on experience with experimental design, buffer and sample preparation, and implementation of instrumentation that is commonly used by experienced researchers while learning and applying the fundamental concepts of protein structure, protein purification, and SDS-PAGE. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):60–68, 2017.
Introduction
Undergraduate biochemistry and biology courses often teach the theoretical concepts of protein purification to include techniques ranging from salt fractionation to liquid chromatography. These principles are then reinforced in laboratory exercises, whereby a quick literature search will yield hundreds of potential experiments for an instructor to choose. Many of these exercises use a single affinity purification step that can simply be accomplished with a gravity flow column. Although these laboratory exercises are very beneficial as introductory purification activities or for larger class sizes, protein purification for functional or structural studies may require multiple purification steps. In a research or industrial setting, protein purification is often accomplished using modern instrumentation like Fast Protein Liquid Chromatography (FPLC). FPLC allows users to monitor several parameters at one time such as UV, pH, and conductance and even allow for multiple columns to be run in tandem minimizing the time needed to obtain pure protein.
Developing a successful purification strategy requires the user to understand the molecular details of their target protein in order to exploit the protein's individual properties (i.e. molecular weight, charge, or hydrophobicity). If an FPLC is employed, users need to apply the theory of the chromatographic column being used to accurately interpret the FPLC chromatogram and corresponding protein detection method, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This is important since protein behavior may differ under native/denaturing or non-reducing/reducing conditions.
Incorporation of an FPLC instrument in undergraduate biochemistry laboratories is not novel 1-3; however, the use of chromatography concepts to interpret FPLC chromatograms and SDS-PAGE gels as a teaching tool for students to learn protein purification has not been reported. The laboratory exercise presented here is a 4-week experiment intended to provide undergraduate students, or potentially first-year graduate students, hands-on experience with an FPLC while emphasizing the theory of size exclusion chromatography (SEC), the theory of ion-exchange chromatography (IEX), and corresponding data interpretation.
The overarching goal of this experiment is to separate individual proteins from a complex mixture [bovine serum albumin (BSA), bovine hemoglobin (Hb), equine myoglobin (Mb) and chicken egg white lysozyme (Lys)] through a multi-step purification strategy based on the differences in their size and isoelectric point (pI). The first part of this experiment uses SEC and SDS-PAGE to challenge student understanding of protein conformation under native and denaturing conditions. Additionally, the students are able to make the connection that both SEC and SDS-PAGE separate based upon size, but SDS-PAGE also separates based upon shape. The latter is seen by comparing samples under non-reducing and reducing conditions. The second part of this laboratory exercise allows the students to operate an FPLC to separate two proteins (BSA and Hb) using IEX chromatography. Students use the pI of the proteins to choose either a cation or anion exchange column and then make the appropriate buffers to elute bound proteins by changing the conductance or the pH toward the pI of the protein. By the end of this exercise, students are expected to have a better understanding of two different protein purification techniques and be able to predict and understand FPLC chromatograms and corresponding SDS-PAGE gels, as well as receive valuable experience with an instrument routinely used in academia and industry.
Materials and Methods
Preparation of Protein Mixture
Approximately 10 mg of each protein [bovine serum albumin (Sigma-Aldrich, St. Louis, MO), equine myoglobin (Sigma-Aldrich), lysozyme from chicken egg white (Sigma-Aldrich), bovine hemoglobin (MP Biomedicals, Santa Ana, CA)] was used for the protein mixture. BSA, lysozyme, and hemoglobin were resuspended in 0.3 mL 50 mM MES, pH 5.8, 300 mM NaCl (at room temp) while myoglobin was resuspended in 1.0 mL 50 mM MES, pH 5.8, 300 mM NaCl (at room temp). All samples were centrifuged at 13,000 × g for 5 minutes to pellet any insoluble material. Soluble protein solutions were mixed together (total volume ∼2.0 mL) to load onto the size exclusion column.
Size Exclusion Chromatography
Sephadex G-75 (Pharmacia Fine Chemicals, Piscataway, NJ) hydrated in 50 mM MES, pH 5.8, 150 mM NaCl was used as the resin for the size exclusion column. The resin was packed into a 1.5 × 50 cm Econo-Column and fitted with an Econo-Column flow adaptor (Bio-Rad, Hercules, CA) because commercially available sephadex columns are very expensive and a sephacryl column will not work for this experiment due to lysozyme's affinity for the resin 4. The column was run on an NGC Quest™ FPLC system (Bio-Rad) at 4 °C while monitoring conductivity and UV at λ280. The isocratic flow rate for the column was 0.2 mL/min while 1.0 mL fractions were collected upon injection of the protein sample.
Preparation for Ion Exchange Chromatography
A single SEC peak fraction was given to each student group for their use on the IEX column. Protein samples were dialyzed against 50 mM MES, pH 5.8, 50 mM NaCl using nominal MWCO of 10 kDa–12 kDa dialysis tubing (Fisher Scientific, Waltham, MA). Tubing was swelled prior to use according to the manufacturer's instructions. Since MES pH is susceptible to temperature changes, students prepared buffers using water that was pre-equilibrated to the appropriate temperature.
Alternatively, dialysis can be bypassed by diluting the SEC fraction with 50 mM MES, pH 5.8 to decrease the salt concentration to match the IEX start buffer (50 mM MES, pH 5.8, 50 mM NaCl). If using this dilution method, err on the side of over-diluting and add the dilution buffer drop-wise to the SEC fraction to prevent protein precipitation. Based upon previous experimental runs, samples applied to the CM or DEAE columns should not exceed 50 mM NaCl. Approximately 3-4 mL of diluted sample can be loaded onto the appropriate ion exchange column.
Ion Exchange Chromatography
Ion exchange chromatography was performed using a 1 mL HiTrap CM Sepharose Fast Flow column or a 1 mL HiTrap DEAE Sepharose Fast Flow column (both from GE Healthcare Lifesciences, Pittsburgh, PA). All samples were loaded onto their respective columns in start buffer (50 mM MES, pH 5.8, 50 mM NaCl) at 1 mL/min. The columns were then washed to a baseline absorbance with 4-5 column volumes of the start buffer. Students then chose to competitively elute the bound protein by increasing salt concentration or by adjusting the pH of the buffer system toward the pI of the protein of interest. For the salt competitive elution, 50 mM MES, pH 5.8, 1 M NaCl was used. Elution by pH depended upon which column was being used. For the DEAE anion column, the elution buffer was 50 mM sodium acetate, pH 4.5, 50 mM NaCl; whereas the CM cation column used 50 mM HEPES, pH 7.0, 50 mM NaCl. For all samples, a gradient of 0-100% elution buffer was applied over 10 minutes at 1 mL/min. One milliliter fractions were collected immediately upon injection to obtain protein that flowed through and eluted.
Students choosing the salt competitive elution used an ÄKTATM FPLC system (GE Healthcare Lifesciences) that is stored at room temperature. This instrument does not have a pH detector. Those students performing the elution by changing pH used an NGC QUESTTM FPLC system (Bio-Rad) that is equipped with a pH sensor and is housed at 4 °C. Data for both the SEC and IEX columns were exported as a CSV file and imported into a spreadsheet program to recreate the chromatograms.
Protein Electrophoresis
Proteins were subjected to separation by 15% SDS-PAGE and visualized by 1% (v/v) 2,2,2-trichloroethanol (TCE)-staining. The TCE in the gel matrix binds to tryptophan residues in protein samples and fluoresces when exposed to UV light, creating a simple visualization method 5-8. Gels were run at 175 V for approximately 45 minutes, removed from casting plates, and placed on a 300 nm transilluminator. Fluorescent bands were visible within 1-2 minutes. Images in this manuscript were captured on a BioRad ChemiDocTM MP system.
Safety Considerations
Unpolymerized acrylamide is a known neurotoxin and gloves were worn at all times while handling gels or gel reagents. While the transilluminator was on, the plexiglass shield was in place. The shield was down when gel images were taken with smart phones.
Timing and Student Participation
Prior to each laboratory day, students were required to complete a pre-laboratory exercise (see supplemental laboratory instructions) that introduced the topics and activities that were covered that day. The instructor, or student laboratory assistant, was responsible for running the SEC column prior to the first laboratory day to allow this experiment to be completed in four days. The students prepared the remaining samples and reagents. Students worked in pairs throughout this exercise and typically finished in approximately three hours with the exception of the third laboratory day, when the students operate the FPLC. With our largest class of eight students, the third laboratory day utilized the entire 4 h. The total amount of time for each group to run the IEX column was ∼1 h. Since we have two instruments near each other, the instructor staggered starting each group by ∼30 min in order to finish all groups in a timely manner.
Results
Our university, primarily an undergraduate institution, has two FPLC instruments for student use, an ÄKTA™ purifier and a BioRad NGC™ system. These are used to support undergraduate laboratory courses and research. This laboratory exercise has been performed in our Biochemical Laboratory Techniques course for three semesters with slight modifications using only the ÄKTA™ purifier. The final version presented here has been performed two times in the Bioinstrumental Laboratory course, which is required for biochemistry majors. Student prerequisites for the laboratory are two semesters of Organic Chemistry, Quantitative Chemical Analysis, Biochemistry I, Introduction to Biochemical Techniques Laboratory, and Bioanalytical Chemistry (a course that emphasizes instrumentation and data analysis for biochemistry majors). The Bioinstrumental Laboratory course has a maximum enrollment of 8 students and meets once a week for 4 h during a 15-week semester.
The current procedure is designed to be completed in four laboratory days. This included time for students to complete the laboratory exercises and the instructor to facilitate in-class discussions. The first day allowed students to familiarize themselves with the FPLC instrument and to compare an SEC chromatogram with the bands obtained from running the corresponding peak fraction on SDS-PAGE gels. For the second day, students reviewed the concepts of IEX chromatography and prepared buffers to run an IEX column. On the third day, students operated an FPLC instrument using an IEX column under the supervision of the instructor. Finally, the fourth day the students analyzed the flow-through and elution fractions from their IEX column by SDS-PAGE to determine if the proteins had been separated.
Prior to the start of the first day the instructor loaded the protein mixture of BSA, Hb, Mb, and Lys on the size exclusion column to obtain a chromatogram (Fig. 1a) and to have samples for student analysis. At the beginning of the first laboratory day, each student was asked to pour two SDS-PAGE gels, a procedure they had performed previously in other laboratory experiments. While the gels polymerized, the instructor showed the students the FPLC instrument, described the flow path of the liquid and the instrument components, and had students practice attaching a column and sample loop. The instructor then handed out the information about the protein mixture used for this experiment (Table 1), led a discussion of SEC, and asked the students to sketch a theoretical SEC chromatogram of the protein mixture.
Size exclusion chromatogram and SDS-PAGE gel of the initial protein mixture. (A) Sephadex G-75 column was run in 50 mM MES, pH 5.8, 150 mM NaCl. Peak fractions are indicated by the letters A-E and were run on the SDS-PAGE gels shown in Fig. 2. The conductance is shown as a line across the middle of the chromatogram and remains constant until the end of the run due to the difference in ionic strength of the sample buffer (300 mM NaCl) and the SEC buffer (150 mM NaCl). (B) SDS-PAGE gel of the protein mixture: Precision Plus ProteinTM Unstained Standards (lane 1) and protein mixture of BSA, Hb, Mb, and Lys loaded onto the SEC column (lane 2). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Most of the students drew a chromatogram that had four peaks, one peak corresponding to each of the proteins, which subsequently led to a discussion about the size difference needed for SEC peak resolution. Approximately half of the students had identical absorbance intensities for all peaks in their predicted SEC chromatogram. The instructor referred the students to the absorptivity column in Table 1 and asked them to consider how this would affect peak height. The students revised their theoretical chromatograms to include only two peaks. The first peak was predicted to encompass both BSA and Hb. Students expected the second peak to contain Mb and Lys and predicted that the height of this peak would be more intense, due to the high absorptivity of Lys.
Then, the instructor gave the students a printout of the actual SEC chromatogram (Fig. 1a) and an SDS-PAGE gel image of the protein mixture (Fig. 1b) that was loaded on the SEC column. The students were surprised that the actual SEC chromatogram differed considerably from their predicted chromatograms. After looking at the SDS-PAGE gel, they quickly observed that there were higher MW proteins above the upper limit of the fractionation range (∼80 kD for sephadex G-75) that would be completely excluded from the resin. These excluded proteins yield an additional peak prior to their predicted BSA/Hb peak. The instructor referred the class to Table 1, and asked if any of the protein properties in the table could contribute to the higher MW proteins. They acknowledged that the larger MW species could potentially be disulfide-linked oligomers of the proteins. Also, students were told the equine Mb is known to form a dimer in solution 9. The instructor told the students that there are protein impurities present in the purchased proteins that are not represented in Table 1, but the focus of this experiment was to separate the four proteins of interest in Table 1 from one another.
Samples from the SEC peak fractions A-E (Fig. 1a) were prepared by the students under reducing and non-reducing conditions, separated by SDS-PAGE, visualized on a transilluminator, and images were captured on their smart phones 8. The SEC chromatogram (Fig. 1a) coupled with student gels (Fig. 2) showed six important concepts. Concept 1: Peak fraction A (Fig. 1a) contained disulfide linked BSA (Fig. 2a, lane 2) that can be reduced to the BSA monomer by the addition of a reducing agent (Fig. 2b, lane 3). This was seen by the oligomer bands, above the 75 kD marker (Fig. 2a, lane 2), that were no longer present under reducing conditions (Fig. 2b, lane 3). The protein bands (Fig. 2b, lane 3) corresponding to the 250 kD and 50 kD markers were impurities present from the manufacturers. The protein migrating at ∼14 kD is Hb. Concept 2: The BSA band under non-reducing conditions (Fig. 2a, lane 2) had a relative mobility (Mr) of ∼50 kD whereas under reducing conditions (Fig. 2b, lane 3) the Mr of BSA was ∼70 kD. This significant mobility shift emphasized that SDS-PAGE separates proteins by both size and shape. Concept 3: SEC peak fractions B and C (Fig. 2a, lane 3 and lane 4 and Fig. 2b, lane 4 and lane 5) had multiple protein bands. These fractions contained both BSA and Hb which was expected due to their similar molecular weights. Therefore, another protein purification technique is needed to further separate these two proteins from each other. The protein band seen at Mr ∼30 kD is an impurity. Concept 4: Under non-denaturing conditions Hb migrated as a ∼64 kD tetramer, composed of two α and two β subunits, which overlaps with BSA monomer, ∼66 kD (Fig. 1a, peaks B and C). However, under denaturing conditions Hb dissociated into individual α and β subunit bands (Fig. 2a, lanes 2-4, Mr ∼ 14 kD). Although difficult to see in Fig. 2, the α and β subunits of Hb do resolve into separate bands, which are more easily seen in Fig. 3d, lane 3. Concept 5: Mb is a non-covalently associated homodimer (Fig. 1a, peak D) that dissociates into a monomer band under denaturing conditions (Fig. 2a, lane 5 and Fig. 2b, lane 6). Concept 6: Lysozyme is a monomer in solution (Fig. 1a, peak E) and migrated on an SDS-PAGE gel as predicted (Fig. 2a, lane 6 and Fig. 2b, lane 7). Although lysozyme has four disulfide bonds, there was not a significant mobility shift seen under reducing conditions, which indicates these bonds do not significantly constrain the shape. As the students analyzed the SDS-PAGE gel from the SEC-fractions, they were able to self-correct mistakes from their previous predictions with the aid of Table 1 and the SEC chromatogram (Fig. 1a). Once they had determined which protein(s) was in each SEC peak fraction, they did not appear to have any issues analyzing the reduced (Fig. 2b) from the non-reduced (Fig. 2a) gel.
15% SDS-PAGE gels of SEC peak fractions. (A) Non-reducing gel of SEC peak fractions (Fig. 1A): Precision Plus ProteinTM Unstained Standards (lane 1), peak fractions A-E (lanes 2-6, respectively). (B) Reducing gel of SEC peak fractions (Fig. 1A): Precision Plus ProteinTM Unstained Standards (lane 1), peak fractions A-E (lanes 3-7, respectively). Lane 2 was intentionally skipped to prevent potential reduction of the standards.
BSA and Hb separation by IEX and corresponding SDS-PAGE gel. Two of the four possible IEX permutations for separating the proteins are shown. One mL samples taken between peaks B and C (Fig. 1A) were loaded on either a HiTrap CM Sepharose column and eluted with a pH gradient (A) or a HiTrap DEAE Sepharose column eluted with a salt gradient (C). The corresponding 15% SDS-PAGE gel for the CM column (B) and DEAE column (D) were loaded in the same orientation: Precision Plus ProteinTM Unstained Standards (lane 1), BSA/Hb mixture before the column (lane 2), flow-through (lane 3), elution (lane 4). For the CM column (B), BSA is present in the flow-through (lane 3) and Hb is eluted off (lane 4). The reverse for the flow-through and protein binding is seen with the DEAE column (D). The individual α and β subunits of Hb are clearly seen in (D) lane 2 and lane 3. Note the protein migrating at Mr ∼30 kD in both (B) and (D) is an impurity present from the manufacturer. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
At the beginning of the second laboratory day, the instructor reiterated the previous result that the BSA and Hb were not separated by SEC. The students were referred back to Table 1 and asked to determine a method to separate these two proteins. Based on the pI differences of the proteins, they were able to discern that IEX chromatography would work best. Since the SEC column was run at a pH of 5.8, BSA has a net negative charge while Hb has a net positive charge, allowing either a cation or an anion exchange column to be used. Students were asked to choose an IEX chromatography method and sketch a chromatogram to illustrate which protein would flow-through the column and which protein would bind to the resin. After finishing their sketch, they had to determine how the IEX start buffer would differ from the elution buffer in order to obtain their predicted chromatogram, whereby they concluded that their elution method would rely upon either changing ionic strength or the pH toward the pI of the bound protein.
The students were also asked how the SEC buffer (50 mM MES, pH 5.8, 150 mM NaCl), that the BSA/Hb mixture is in, might affect running their samples on an IEX column. They deduced this ionic strength may compete with a protein binding to the column and that they would need to lower the salt concentration either through dialysis or dilution. This salt dilution step has been successfully performed through dialysis against the IEX start buffer or directly diluting the SEC fraction with buffer that had 0 mM NaCl (50 mM MES, pH 5.8). The students then prepared an elution buffer that would allow them to elute the protein based on the ion exchange resin they chose.
The third laboratory day was devoted entirely to having the students operate the FPLC to separate BSA from Hb. Each student group repeated the procedure of connecting the column and the sample loop, using their notes from the first laboratory day, which allowed the instructor to evaluate their familiarization with the instrument. The students then operated the FPLC under the supervision of the instructor, collecting fractions of the flow-through and the elution. After each student group had finished its run, they re-equilibrated the FPLC system in water and disconnected the column and sample loop so the next student group could use the instrument. All four permutations (using a cation or anion exchange column and eluting with either salt or pH) can be used to successfully separate the BSA from Hb. Student chromatograms for two of the four permutations, carboxymethyl (CM) cation exchange with pH elution and diethylaminoethyl (DEAE) anion exchange with salt elution, are shown in Figs. 3a and Fig. 3c, respectively. As expected, there are two protein peaks, one for the flow-through and a second after the elution gradient is applied. Students were instructed to record observations about the color of their fractions and to make SDS-PAGE samples of the protein mixture before the column, a peak fraction from the flow-through, and a peak fraction from the elution. If there were time constraints, sample preparation for the gel was done at the beginning of the next laboratory day.
On the fourth laboratory day, the students ran an SDS-PAGE gel of their IEX samples from the previous laboratory day. Student gels (Figs. 3b and Fig. 3d), corresponding to the ion-exchange chromatograms (Figs. 3a and Fig. 3c) show the IEX columns successfully separated BSA from Hb. When students used the CM column, BSA did not bind to the column and was present in the flow-through (Fig. 3b, lane 3) while Hb bound to the column and was eluted off by increasing the pH (Fig. 3b, lane 4). In contrast, when using the DEAE column, Hb does not bind and is present in the flow-through (Fig. 3d, lane 3) while BSA does bind and can be eluted by increasing the salt concentration (Fig. 3d, lane 4). These results were as expected based on the starting buffer pH and the theoretical pIs of the proteins.
Student Feedback
After the in-class discussion of the principles of SEC and SDS-PAGE and comparing the SEC chromatogram to their gels, students were able to differentiate protein structure changes under native and non-native conditions as seen by oligomer dissociation under denaturing conditions and mobility shifts under reducing conditions. These observations led to a discussion of why the units of Mr are used instead of molecular weight when describing a gel. When first introduced to the instrument, the students were intimidated by all of the tubing. Previously, Bellin et.al. reported a lack of student confidence when using an FPLC without a prior demonstration. Therefore, we had our students practice attaching the columns and sample loops at the beginning of the first laboratory day and then again on the second laboratory day to help with their familiarity and overall confidence. These multiple practice periods benefitted the students. They realized that if they understood where the components were located and the flow path of the liquid, the instrument was fairly user friendly. A follow-up survey, discussed below, indicated that greater than 85% of students either agree or strongly agree that they felt more confident using the instrument and all students agree or strongly agree with being able to describe how to attach a column and sample loop. Finally, some students commented that they enjoyed being able to easily monitor whether BSA or Hb bound to the IEX column or flowed through based on the color of the fractions collected since the Hb fractions have a reddish tint and BSA is colorless.
One pitfall encountered was that the SDS-PAGE gels occasionally leaked during the casting procedure. To avoid a disruption in pacing of the exercise, on the first day each student was asked to pour two gels, providing backups as needed. On the second or third laboratory day there was time for students to re-pour a gel in preparation for the fourth laboratory day. Two additional issues encountered were either the protein not binding or not eluting from the IEX column. Students could easily find their mistake for either scenario by following the conductance or pH throughout their run. If the protein did not bind to the column it was due to insufficiently diluting their SEC peak fractions to match or be below the IEX start buffer salt concentration. If the protein did not elute from the column after the full gradient was applied, it was due to mistakes preparing the elution buffer. We have found that successful buffer preparation can be accomplished by thorough pre-lab questions and an in-class discussion on the second laboratory day before students prepare their buffers.
The most recent group of eight students who completed this laboratory exercise were given a Likert scale survey to self-evaluate their understanding of the following topics (the numbers in parenthesis correspond to the percentage of students who either strongly agree (S.A.) or agree (A.) with the statement): A. Setting up an FPLC instrument (S.A. 62.5%, A. 37.5%), B. Ability to interpret SEC chromatograms (S.A. 37.5%, A. 62.5%), C. Ability to interpret IEX chromatograms (S.A. 50%, A. 50%), D. Better understanding of how to interpret SDS-PAGE gels (S.A. 50%, A. 0%), E. Ability to develop a protein purification strategy using an FPLC (S.A. 75.0%, A. 12.5%). Overall, the students felt more confident using an FPLC and in their abilities to analyze SEC and IEX data. Interestingly, only half of the students responded that they had a better understanding of how to interpret SDS-PAGE gels and the other half of the students were neutral. This is likely due to the fact that using SDS-PAGE is a skill presented in earlier prerequisite courses while half of the students surveyed in this course were currently performing undergraduate biochemistry research that routinely uses the technique.
Lastly, our university uses the full length ACS-Biochemistry Exam as an assessment tool at the end of the Biochemistry II lecture course, offered concurrently with the laboratory course in which this exercise was completed. As a method to track student learning, we compared student responses to questions that directly correspond to topics covered in this laboratory exercise. The responses of those who performed this exercise and those who did not were compared. The average score, based on these questions, for students who performed this exercise was 86.7% (n = 10) compared to 59.0% (n = 13) for those who did not. These data were collected over three semesters.
Discussion
A recommended laboratory skill within a biochemistry and molecular biology curriculum is the ability to isolate and characterize proteins through the use of chromatography and electrophoretic techniques 10. Although protein purification is a commonly discussed biochemistry topic and numerous laboratory experiments involve protein purification strategies, few of these laboratory exercises allow students to directly use an FPLC and those that do, integrate FPLC use as a smaller piece of a larger multi-week project 1, 2. The laboratory experiment presented here is designed to be an initial exposure to using an FPLC instrument while emphasizing the fundamentals of protein chromatography and electrophoretic techniques. This exercise is ideally suited for undergraduate institutions with small biochemistry or molecular biology laboratory classes, or as an activity for first-year graduate students needing to gain experience with an FPLC. Instructors could use this exercise to strengthen student skill in interpreting protein purification data (i.e. chromatograms and SDS-PAGE gels) prior to performing a multi-week capstone or research project.
The proteins in this experiment were chosen based upon their properties of molecular weight, pI, oligomeric state under native and non-native conditions, and, most importantly, commercial availability and cost. This experiment was designed to initially use the SEC column to separate two of the four proteins and then have students use IEX to separate the remaining two proteins. Equine myoglobin was chosen because its molecular weight is similar to that of lysozyme, but it dimerizes in solution 9. This latter property allowed for complete separation from lysozyme by SEC and reinforced the requirement for at least a doubling of molecular weight for separation by SEC. Additionally, having two heme proteins (Hb and Mb) forced the students to use the data provided in Table 1 and the SEC chromatogram, Fig. 1a, to distinguish the proteins based upon solution-state instead of identifying based solely on the color of the SEC fractions.
The SEC chromatogram of the protein mixture served three purposes. First, it was used with a corresponding SDS-PAGE gel during the first laboratory day to illustrate the difference between protein conformations under native and non-native conditions. Second, a laboratory follow-up question (see supplemental laboratory instructions) asked students to use the chromatogram and estimate the bed volume of the SEC column based upon the difference between Vo (void volume), estimated by the BSA oligomer peak (Fig. 1a, peak A), and Vi (inclusion volume), estimated by the change in conductance (Fig. 1a). The change in conductance was due to the initial sample being applied in 300 mM NaCl whereas the column was equilibrated and run in 150 mM NaCl. This conductance change also reinforced the principles of SEC by allowing students to observe that very small molecules completely penetrate the stationary phase. Finally, the SEC chromatogram was used for another follow-up question that asked the students to create standardization plots by graphing the log10 MW against Ve/Vo (elution volume/void volume) and predicting where a protein of known molecular weight would elute. An example of a student plot can be found in the supplemental laboratory instructions.
Following analysis of the SEC chromatogram and SDS-PAGE gels, students observed that the BSA and Hb were not separated. Based upon the pH of the SEC column buffer and the pIs of BSA and Hb, either cation or anion exchange chromatography could be used to separate these two proteins, which challenged the students to develop an appropriate protocol. After completing this second chromatographic step, the student data showed that all four proteins were separated from one another. Students gained hands-on experience with experimental design, sample preparation, making buffers, and operating an FPLC over the course of this exercise.
Although one feature of this laboratory exercise is to expose students to using an FPLC, not all universities have an FPLC available for student use. In the absence of having this instrument, instructors may still use this exercise to emphasize the concepts of protein conformation under native and non-native conditions and protein separation. A simple modification of the exercise using the pre-lab questions and concept activities implemented in this exercise to challenge students to sketch chromatograms and predict protein migration on SDS-PAGE gels could be used if the instrument is unavailable.
Conclusion
This exercise is a hands-on approach to introduce students to operating an FPLC instrument, routinely used in academia and industry, to emphasize the principles of protein purification through the use of SEC and IEX chromatography. Additionally, students are exposed to how protein conformations change under denaturing/non-denaturing or reducing/nonreducing conditions by comparing SEC chromatograms to corresponding SDS-PAGE gels.
Acknowledgments
The authors thank the Armstrong State University College of Science and Technology for their support through Summer Research Session Grants. This work was further supported by the National Science Foundation's STEP Program under Award No. DUE-0856593. Any opinions, findings, and conclusions or recommendations expressed in this manuscript are those of the authors and do not necessarily reflect the views of the National Science Foundation.
