Laboratory Exercise An Adaptable Investigative Graduate Laboratory Course for Teaching Protein Purification
Christopher W. Carroll† Lani C. Keller‡*
From the †Department of Cell Biology, Yale University, New Haven, Connecticut 06520, ‡Department of Biological Sciences, Quinnipiac University, Hamden, Connecticut 06518
Abstract This adaptable graduate laboratory course on protein purification offers students the opportunity to explore a wide range of techniques while allowing the instructor the freedom to incorporate their own personal research interests. The course design involves two sequential purification schemes performed in a single semester. The first part comprises the expression and purification of a recombinant GFP-binding protein from E. coli. The student-purified GFPbinding protein is then used in the second part of the course to immunoprecipitate GFP-tagged proteins, and their potential interacting partners, from cell or tissue extracts. As an example, we describe the immunoprecipitation of GFP-tagged proteins from Drosophila melanogaster
larval extracts that are homologous to proteins implicated in human diseases, followed by western blotting to examine student experimental outcomes. However, the widespread availability of GFP-fusion proteins in diverse organisms enables researchers to tailor the second part of the course to their specific research programs while maintaining the flexibility to engage students in active learning. Student evaluations indicate a genuine excitement for research and in depth knowledge of both the techniques C 2014 by The performed and the theory behind them. V International Union of Biochemistry and Molecular Biology, 42(6):486–494, 2014.
Keywords: integration of research into undergraduate teaching; laboratory exercises; new course development; green fluorescent protein; molecular biology; Drosophila; protein purification
Introduction Teacher-scholars are committed to providing high-quality education while maintaining an active research program. The teacher-scholar model promotes the idea that students engaged in research gain a deeper understanding of material presented in traditional classroom settings, and have an enhanced ability to integrate and synthesize information [1]. For teaching and research to be mutually beneficial, it is essential to combine them synergistically [2]. Here, we outline a semester long laboratory course in protein purification that enables teacher-scholars to engage students in research-oriented, active learning while performing experi-
*Address for correspondence to: Department of Biological Sciences, Quinnipiac University, Hamden, Connecticut 06518, USA. E-mail: [email protected] Received 15 April 2014; Accepted 7 September 2014 DOI 10.1002/bmb.20827 Published online 21 October 2014 in Wiley Online Library (wileyonlinelibrary.com)
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ments that can be tailored to the research interests of the teacher-scholar. The use of green fluorescent protein (GFP) fusion proteins in laboratory classes is ideal for teacher-scholars. Educators have effectively employed GFP as a tool to teach the fundamental principles of protein purification in vitro, and it has been used to involve students in structurefunction studies through site-directed mutagenesis and spectrophotometry [3–5]. However, these approaches do not allow the instructor the flexibility to incorporate their own research interests into the course material. The course described here provides an opportunity for students to engage in research-oriented learning, as recommended by the National Research Council, while contributing to the research program of the instructor [6]. Moreover, the widespread use and availability of GFP-fusion proteins in virtually all academic research settings allows students and instructors alike a vast array of choices for material sources and biological problems to explore. Thus, students are empowered to take ownership of their projects while teacher-scholars are provided the flexibility to incorporate their own research interests into the course.
Biochemistry and Molecular Biology Education
The course is broken down into two integrated protein purification procedures. The first involves the expression and purification of a high affinity GFP-binding protein from bacteria. The GFP-binding protein consists of a fragment from a single-domain camelid antibody engineered to bind GFP with high affinity [7]. The sequence of the GFP-binding protein is cloned downstream of a 6-Histidine epitope tag in a bacterial expression vector, permitting purification by Nickel-affinity column chromatography using standard biochemical techniques. Single chain antibodies hold great promise as potential therapeutics and expose students to an emerging class of reagents that are increasingly important in academic and industrial research settings [8]. The subsequent procedure incorporates active learning as a method of exploration. The student-purified GFP-binding protein is coupled to a solid support and used to isolate a GFP-tagged fusion protein and its interacting proteins [3]. Described here is a procedure for the immunoprecipitation of GFP-fusion proteins from Drosophila larval extracts that are prepared from freely available reagents described in the GFP protein trap database (flytrap.med.yale.edu) [9]. Early in the semester, student research teams choose a Drosophila homolog of a human disease protein that they are interested in studying. They are then responsible for designing immunoprecipitation experiments to test hypotheses about protein localization, protein interaction partners, and ultimately the molecular mechanisms leading to disease. Student feedback demonstrated excitement about research, and a positive learning environment.
Course Synopsis The described course has been offered since 2012 and exists as an updated version of a long-running protein laboratory techniques course, which is one of five core courses required for all students in the Molecular and Cell Biology Master’s program at Quinnipiac University. Enrollment is usually between 30 and 45 students each fall, which are split into two or three sections with an average of 12–15 students per section. Acceptance into the Molecular and Cell Biology or Biomedical Science graduate program is required and graduate level biochemistry is a prerequisite. This laboratory class is taught in an interactive, hands-on learning format as a semester-long 4-hr/week introductory course. It could easily be adapted, however, to be delivered as an accompanying laboratory within upper-level undergraduate courses in biochemistry, molecular biology, or cell biology. Teams of two to three students are created on the first day of the course and act as collaborative student research teams throughout the semester. Each research team works in a technology pod with a computer providing continual access to the Internet, on-line lecture material, laboratory protocols, and all necessary programs and databases such as Excel, FlyBase (http://flybase.org/), and the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov/). These
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collaborative research teams serve to enhance learning by encouraging students to apply their knowledge, problemsolve and actively engage in concurrent data analysis. Lecture material was provided exclusively through on-line lectures, and students were expected to complete assigned reading from [10] and watch the on-line course material before class to allow for full participation in problem- solving and hands-on experimentation. The required text [10] provides an entirely self-contained practical and simple guide to protein purification. Although slightly out of date, this text is a tried and true reference that delivers easy to understand step-by-step procedures with clear illustrations for the majority of routinely used techniques in protein purification. This reference text was supplemented with current materials such as primary literature, manufacturer-supplied protocols, and in-depth explanations of contemporary topics such as nanobodies. On-line lecture material was assembled from a variety of these resources. A graduate assistant who previously took the course and an instructor who actively conducts research with Drosophila did all prelaboratory preparation. Stock solutions were made prior to each experiment to save time and allow for maximal student experimentation. Each research team was, however, required to prepare working solutions from provided stock solutions. Laboratory experiments were separated into two multipart sections with the goals of: 1) purifying a GFP-binding protein from E. coli and 2) using this GFP-binding protein to immunoprecipitate a GFP-tagged protein from Drosophila extracts (Table 1). Laboratory exercises were designed to incorporate a large range of protein purification techniques from cell lysis and affinity purification to immunoprecipitation and protein visualization by SDS-PAGE and immunoblotting. Importantly, while the first part of the course was standardized for all student research teams, each team selected a unique target protein for immunoprecipitation based on the research interests of the instructor and the students themselves in the second part of the course. Students were assessed through weekly in-class learning assessments, two laboratory exams based solely on the theory and practical applications of the material, class participation, and a laboratory notebook. In addition, each student research team prepared an oral presentation at the end of the course in which they provided the rationale for choosing a particular GFP-fusion protein for immunoprecipitation, candidate interacting proteins, and potential mechanisms by which mutations in the human homolog of the chosen protein could cause the associated human disease. The essential learning outcomes for the course included critical thinking and reasoning, scientific literacy, and quantitative reasoning.
Expenses Shared resources and supplies were crucial for designing and running this course, and all basic laboratory equipment
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TABLE 1
Week-by-week summary of laboratory experiments
Part I: GFP-Binding Protein Purification from bacteria 1
Bacterial cell lysis
2
Nickel affinity purification
3
Bradford protein assay
4
SDS-PAGE
5
Coupling GFP-Binding protein to beads
the instructor hoped the students would learn. It is important to define a set of essential questions that have the potential to motivate students to pursue facts and understand the basis of biological problems introduced within a course [11]. The essential questions that guided this course are: What makes the study of proteins more complicated and demanding than the study of nucleic acids? How have biotech companies developed methods to produce purified proteins like insulin? How could you discover all the proteins within a cell that interact with the Huntingtin protein, and why is this information important? How have advances in protein expression and purification impacted individuals and society?
Part II: Immunoprecipitation of GFP-tagged proteins 6
Lysis of samples expressing GFP-tagged protein
Laboratory Experiments
7
Immunoprecipitation of GFP-fusion proteins
Part I: GFP-Binding Protein Purification
8
SDS-PAGE
Prelaboratory Preparation of Bacterial Cells Expressing GFP-Binding Protein
9
Immunoblotting
10
Student Presentations
including a sonicator, a spectrophotometer, centrifuges, SDS-PAGE, and western blotting apparatuses were already on site. All chemicals and other reagents were either borrowed or purchased using funds specifically set aside for the Molecular and Cell Biology program within the Department of Biological Sciences at Quinnipiac University. Initial costs can be substantial, running anywhere between $500–2500; however, costs are considerably cheaper in subsequent course sessions because most reagents can be used for several semesters. Creativity and the simple action of reaching out to nearby institutes and professors goes a long way in providing the necessary reagents to fund a laboratory course like the one described here. The main costs associated with this course include desalting columns ($250), anti-GFP antibody and HRP-conjugated secondary antibody ($500), SimplyBlueTM SafeStain ($122.61), Ni-NTA agarose ($259- this can be used for many years for a variety of protein purification techniques), chemicals including imidazole, ampicillin, PMSF, NaCl, BME, Ponceau, and lysozyme ($375), NBT/ BCIP substrate solution ($124.44- can use for years), Bradford reagent ($127.00- can use for years), disposable columns ($210.00- enough to be used for many years), NHSActivated Agarose slurry ($210.00), and protein concentrators ($151.00- however this step can be easily substituted for dialysis which is significantly cheaper but requires treatment of samples in dialysis tubing with a high molecular weight osmotic like polyethylene glycol to initiate protein concentration).
Course Essential Questions Essential questions for the course were presented on the first day of class, within the syllabus and elaborated what
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BL21-CodonPlus (DE3)-RIPL cells (a bacterial strain carrying the gene for expression of the T7 polymerase and supplemented with extra copies of the arg, ile, leu, and pro tRNA genes; Fisher #NC9122855) were transformed with a pET28 6-HIS expression vector with the GFP-binding protein previously cloned in (developed for academic purposes only, available from corresponding author by request). Bacteria were plated on LB-agar plates (Sigma #L7025) supplemented with Kanamycin (Sigma #K1377). A single colony was chosen the next day and added directly to a 50 mL culture of 2X YT (2X Yeast Extract and Tryptone; Sigma #Y1003) media supplemented with 50 mg/mL of Kanamycin for overnight growth. This culture was then diluted back and grown in 2 L of 2X YT media at 37 C until an optical density of 0.8–1 was reached by spectrophotometric analysis at 600 nm. Expression of the GFP-binding protein was then induced with 0.1 mM IPTG (Sigma #I5502) and cultures were moved to 23 C. After growth for 20 hr, cells were then spun down, washed with 13 phosphate buffered saline (purchased as 103 PBS, Invitrogen #70011-044), pelleted in 50 mL conical tubes (BioExpress #C-3394-4), and stored at 270 C for future use.
Laboratory Experiment 1: Bacterial Cell Lysis During this laboratory, students learn the basics of cell lysis and use multiple methods to disrupt E. coli and liberate the highly expressed, soluble GFP-binding protein. Prior to beginning laboratory experiments, it is essential that all students are comfortable micropipetting. Instructors should therefore incorporate a pipetting activity the week prior to starting the purification scheme. Each student research team receives one aliquot of pelleted bacteria expressing the GFP-binding protein. Throughout the purification it is important to keep all samples on ice. Each research team is responsible for preparing 20 mL of lysis buffer [13 PBS with 300 mM NaCl
Laboratory Course for Teaching Protein Purification
(Sigma #S3014); 20 mM imidazole (Sigma #56750); 1 mM PMSF (Sigma #P7626 or Thermo Scientific #88660); 100 mg/mL lysozyme (Thermo Scientific #89833)] and gently resuspending their cell pellets. PMSF is a cytotoxic protease inhibitor and should only be handled with proper personal protection equipment such as gloves and laboratory goggles. Students then homogenize their sample by sonication on ice. Sonication produces dangerous high frequency sound waves so earplugs should be required while sonication is in progress. Each student research team then removes 100 mL of their homogenized sample and adds it to a microcentrifuge tube (BioExpress #C-3260-2) labeled “Whole Cell Lysate.” The remaining lysed bacterial extract is centrifuged at 10,000 3 g at 4 C for 20 min to generate a cleared lysate that contains the soluble GFP-binding protein. After centrifugation each student research team then removes 100 mL of supernatant and adds it to a microcentrifuge tube labeled “Cleared Lysate.” The remaining supernatant is saved for subsequent purification steps in a 50 mL tube labeled “Soluble Fraction.” The pellet can be discarded, although care should be taken to ensure that proper institutional recombinant DNA protocols are followed. At the end of this experiment, all student research teams should have three tubes stored at 220 C: (1) 20 mL Soluble Protein Fraction, (2) 100 mL Whole Cell Lysate, and (3) 100 mL Cleared Lysate.
Laboratory Experiment 2: Nickel-Affinity Purification of GFP-Binding Protein In this experiment, students acquire skills in affinity chromatography through a tried and true 6-HIS purification scheme. Each student research team starts this laboratory exercise by thawing their “Soluble Protein Fraction” and putting it on ice. Nickel affinity matrix (Ni-NTA, Qiagen #30210) is then prepared by pipetting 1.5 mL of 50% NiNTA slurry into a microcentrifuge tube and washing it three times in wash buffer (13 PBS 1 20 mM imidazole). The remaining “Soluble Protein Fraction” is then combined with the washed Nickel affinity resin and gently agitated at 4 C for 1 hr to allow binding. The cleared lysate and nickel affinity matrix mixture is then added to a plastic column (Thermo Scientific #29924) and 20 mL of flow through is collected and saved into a labeled 50 mL conical tube, of which 100 mL is transferred to a microcentrifuge tube labeled “Flow Through.” The chromatography column is washed three times with 5 mL of wash buffer to remove all non-specific binding of proteins to the affinity matrix. The 6-HIS-tagged GFP-binding protein is then eluted from the column by collecting ten 1 mL fractions with 13 PBS containing 500 mM imidazole. Note that high concentrations of imidazole can alter the pH of the solution; it is therefore critical to dilute the imidazole in the elution buffer from a 1 M stock that has been adjusted to pH 8.0. It is important for students to think critically about the process of affinity chromatography and to hypothesize where their 6-HIS-
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tagged GFP-binding protein will elute within the collected elution fractions. All elution fractions are labeled and stored at 220 C for further analysis.
Laboratory Experiment 3: Bradford Assay to Determine Protein Concentration of GFP-Binding Protein In this experiment, students learn how to create a standard curve demonstrating the relationship between absorbance at 595 nm and known concentrations of bovine serum albumin. Any available spectrophotometer capable of reading absorbance at 595 nm may be used for this exercise. Each student research team is responsible for creating their own standard curve within their technology pod, which is then used to extrapolate accurate protein concentrations of their GFP-binding protein. To create standard curves, students first must make a wide range of BSA concentrations (0, 125, 250, 500, 700, 1000, 1500, and 2000 mg/mL) from a 2.0 mg/mL stock solution (Sigma #P0834). Students are responsible for making all working solutions throughout the course to reinforce their quantitative reasoning skills. Once the standard curve BSA solutions are made, 10 mL of each is individually mixed with 500 mL of 13 Bradford reagent prepared according to manufacturer guidelines (Thermo Scientific #23236). The Bradford reagent, originally a red color, turns blue when bound to BSA, thus providing students with a means to qualitatively assess the protein concentration of their elution fractions prior to spectrophotometric analysis. The absorbance at 595 nm of each BSA solution is then determined by spectrophotometry and a standard curve (including line equation and R2 value to determine pipetting accuracy) is immediately created in Excel. Next 10 mL of each elution fraction is added to 500 mL of the Bradford reagent to determine the concentration of each elution fraction. Qualitative comparisons are made by eye before quantitatively measuring the absorbance of each protein fraction. The student-generated standard curves allow students to accurately determine the protein concentration of each of their protein fractions.
Laboratory Experiment 4: SDS-PAGE to Visualize Purification of GFP-Binding Protein In this exercise, students learn how to load, run, stain, and R analyze SDS-PAGE gels. Nu-PAGEV gels (Invitrogen #NP0321BOX) with MES SDS buffer (Invitrogen #NP0060) are utilized because full electrophoresis is complete within 30 minutes. Each student research team prepares the following samples by addition of sample buffer (Invitrogen #NP0007) supplemented with b-mercaptoethanol (Sigma #M3148) to break disulfide bonds: 20 mL “Whole Cell Lysate”, 20 mL “Cleared Lysate”, 20 mL column flowthrough, and 20 mL of elution fractions 1–6. bmercaptoethanol is hazardous and should only be used in the hood with proper personal protection equipment. Samples are then denatured by at 95 C for 10 min immediately before loading the gel. While the samples denature, Nu-
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Biochemistry and Molecular Biology Education R gels are prepared by rinsing them under water, PAGEV removing the tape and gel combs. Samples and 10 mL of prestained protein ladder (Invitrogen #10748-010), are then loaded and gels are run at 200 V for 30 min. It is important for students to ensure that there is a measurable current and to record in their laboratory notebooks what that current is. Students running gels that are not properly inserted into the apparatus may not show any current, while improper dilution of running buffer may cause the current to be higher than expected. Running the gels at a constant voltage of 200 V, should give an expected current of 110–125 mA at the start and 70–80 mA near the end. If students are unfamiliar with protein gel loading, it is important to have a practice gel and extra loading dye available. Gel-loading tips (Fisher #05–408-153) can also aid in the students’ ability to properly load protein gels. After electrophoresis, gels are carefully removed and stained with SimplyBlueTM Safestain (Invitrogen #LC6060) according to manufacturer guidelines. Students analyze their gels to investigate the purification of the soluble GFPbinding protein from cleared bacterial lysate (Fig. 1). Comparisons between student research team gels and between hypothesized results and obtained results are a great way to enhance critical reasoning skills. Results obtained by Bradford assay and by SDS-PAGE can also be compared to investigate purity of the samples. Each student research team must determine the three elution fractions with the highest concentration of purified GFP-binding protein and save these samples at 220 C for subsequent experimental steps.
Laboratory Experiment 5: Preparation of Purified GFPBinding Protein and Coupling to Agarose Beads The covalent coupling of purified and concentrated GFPbinding protein to a monovalent matrix, such as agarose beads, allows for subsequent fast and efficient one-step isolation of GFP fusion proteins from a sample of the instructor’s choosing. It is first important to remove the high concentrations of imidazole and to concentrate the GFPbinding protein to ensure efficient coupling. To provide students with additional column chromatography experience, size exclusion column chromatography is incorporated through the use of PD-10 desalting columns (Fisher #45000-148). Traditional dialysis may also be used to remove imidazole, although desalting columns provide a fast alternative. The advantages and disadvantages of choosing either dialysis or size exclusion chromatography should be discussed. To conduct desalting by size exclusion chromatography, each student research team must equilibrate the PD-10 column with equilibration buffer (13 PBS). The top three elution fractions from the previous laboratory exercise with the most protein according to Bradford and SDSPAGE are then combined and added to the column and flow-through is discarded. The soluble, desalted GFPbinding protein is then eluted and collected by the addition
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FIG 1
Student SDS-PAGE gel stained with SimplyBlueTM Safestain to visualize nickel-affinity purification of GFP-binding protein from E. coli. Whole cell lysate and the cleared lysate show large numbers of proteins expressed in E. coli with a band at 19 kD representing the highly expressed GFP-binding protein. Depletion of the GFP-binding protein in the column flow through indicates successful binding of the 6-HIS GFPbinding protein to the Nickel affinity chromatography column. Elutions 1–5 demonstrate purification of the GFP-binding protein with the highest expression in elution one and a tapering off there after. Arrow indicates successful purification of 6HIS-tagged GFP-binding protein.
of 3.5 mL 13 PBS. At this point the imidazole is not yet eluted and therefore remains within the matrix of the column. Following size exclusion chromatography, a 20 mL aliquot of the desalted GFP-binding protein is saved while the remaining protein is concentrated by centrifugation in Pierce Protein Concentrators with a 9 K Molecular Weight Cut Off (Thermo Scientific #89884A). A quick qualitative Bradford assay is conducted to determine if the GFPbinding protein concentration is in fact increased (sample after concentration should have a higher concentration of GFP-binding protein and therefore will be more blue than prior to centrifugation). Student research team then takes their remaining concentrated sample and covalently couples it to NHS-activated agarose beads by incubation for 20 min at room temperature with gentle rocking. Beads are prewashed with water three times prior to student use. To stop the coupling reaction, 500 mL of 100 mM Tris, pH 7.5 is added for 15 min and then removed by centrifugation. A quick Bradford assay can again be used to ensure that the purified GFP-binding protein is coupled to the beads by comparing a sample before coupling to the supernatant after coupling (supernatant after coupling should be less blue than starting sample because the purified GFPbinding protein is now coupled to the agarose beads).
Laboratory Course for Teaching Protein Purification
Reintroducing the Bradford assay multiple times throughout the course reinforces the importance of following a protein through a protein purification procedure and allows students to make hypotheses at many points throughout the protocol. The coupled agarose beads are stored at 4 C and are washed once in 13 PBS prior to being used in immunoprecipitation experiments.
Part II: Immunoprecipitation from Drosophila The second half of the course allows students to take more ownership of their project and engage in active and collaborative learning within their research teams. Teach student research team selects a human disease of their interest to research. They are required to determine what genes and proteins are disrupted or involved in causing the disease state and establish which genes have homologues in Drosophila. The student research teams use FlyBase (http://flybase.org/), the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), and FlyTrap (http:// flytrap.med.yale.edu/) databases to learn about their genes/ proteins, find publicly available GFP-tagged lines, and make predictions about protein localizations, expression levels, and protein-protein interactions. Student research teams then design and perform immunoprecipitation experiments using their purified GFP-binding protein from the first half of the semester. The basic immunoprecipitation procedure was similar between research teams and is described in detail below, however the experimental details and hypotheses that each research team asked were unique. This process promotes active learning, reading, writing, discussion, and problem-solving skills and culminated in the oral presentation of their work to the class. The presentations were required to include their experimental procedures and results, descriptions of the known molecular mechanisms causing the disease in humans, and explanations of how the use of model organisms may help in understanding and potential treatment of their particular disease.
Laboratory Experiment 6: Lysis of Drosophila Larva Expressing GFP-tagged Proteins The second part of this laboratory course focuses on the isolation of GFP fusion proteins from the model organism Drosophila, although it is important to note that instructors may employ any GFP-expressing model organism or in vitro system that is relevant to their research interests. In the laboratory exercise described here, students attain hands-on experience working with Drosophila and compare and contrast cell lysis procedures used previously for bacteria to those used for Drosophila larva. The instructor must preorder Drosophila lines expressing GFP-tagged proteins based on student research team interest from FlyTrap (http://flytrap.med.yale.edu/) and ensure that each team has 50 Drosophila larva or adults depending on their experimental design. The GFP-expressing larva are collected and frozen at 280 C for 10 min to increase lysis efficiency.
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Larva are then thawed and exposed to 200 mL of fly lysis buffer (1X PBS; 150 mM EDTA; 0.1% NP-40; 10% glycerol; 1 mM PMSF) before homogenization in an microcentrifuge tube with a Kontes motorized tissue grinder (Fisher #K749540-0000) and RNase-Free disposable pellet pestles (Fisher #12–141-364). Homogenized Drosophila larva expressing GFP fusion proteins are then centrifuged at high speed for 15 min at 4 C to pellet nonsoluble proteins. The supernatant containing all soluble proteins are saved and stored at 220 C for the following week.
Laboratory Experiment 7: Immunoprecipitation of GFP Fusion Proteins Immunoprecipitation is a technique for isolating a protein from a complex mixture using an antibody that specifically binds that particular protein. In this laboratory, the GFPbinding protein (nanobody acting as an antibody) is coupled to NHS-activated agarose beads (Thermo Scientific #26200) to precipitate the GFP fusion protein (antigen) from Drosophila lysate. Purified GFP can act as a positive control to ensure that coupled beads have the capacity to precipitate GFP, if instructors have access to it. Each student research team first thaws their cleared Drosophila lysate and removes a 10 mL aliquot, which is labeled “input.” The “input” is mixed directly with sample buffer and stored at 220 C. The cleared Drosophila lysate is then added to the agarose beads coupled to GFP-binding protein and incubated at 4 C for 1 hr with gentle rocking. The 1-hr incubation period allows for class discussion of immunoprecipitation procedure and predictions of expected outcomes. The GFP-binding protein on the agarose beads should bind with high affinity to any GFP-tagged protein within the Drosophila larval lysate. After the 1 hr binding step, agarose beads are centrifuged down and a 10 mL aliquot of the supernatant is removed and labeled “supernatant.” Sample buffer is added directly to the “supernatant” and stored at 220 C. Samples are then recentrifuged to pellet agarose-beads and washed three times with 13 PBS. During washes, care must be taken to avoid pipetting beads, which will be bound to GFP-tagged proteins and any interacting proteins. Pelleted beads are then resuspended directly in 20 mL of sample buffer and stored at 220 C.
Laboratory Experiment 8: SDS-PAGE and Transfer onto Western Blot for Immunoblotting Each student research team prepares the following samples for SDS-PAGE: 1) prestained protein ladder, 2) purified GFP “input” as a positive control, 3) “supernatant” after immunoprecipitation of purified GFP, 4) agarose beads after immunoprecipitation with purified GFP, 5) prestained protein ladder, 6) Drosophila cleared lysate “input”, 7) “supernatant” after immunoprecipitation, and 8) agarose beads after immunoprecipitation with GFP-tagged protein R gel bound. Samples are then loaded and in a Nu-PAGEV and exposed to 200 V for 30 min as described above. Once again, it is important for students to ensure that there
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Biochemistry and Molecular Biology Education is a measurable current and to record in their laboratory notebooks what that current is. After electrophoresis, gels are carefully removed and traditional western blot transfer R is set up using NuPAGEV transfer buffer (Invitrogen #NP0006-1) and pre-prepared nitrocellulose filter paper sandwiches, 0.45-mm pore size (Invitrogen #LC2006). The use of a prestained ladder allows for students to visualize effective transfer onto nitrocellulose membrane. To investigate total protein transferred onto membrane, students perform Ponceau (Sigma #P3504) staining of the nitrocellulose membrane. Students should make predictions about what proteins bind to the nitrocellulose versus those proteins that will be visible after western blot analysis. After Ponceau staining, student observations are made and nitrocellulose membrane is washed with distilled water, dried, and stored at room temperature.
Laboratory Experiment 9: Immunoblotting and Detection of Immunoprecipitated GFP-tagged Protein from Drosophila Prior to starting this laboratory experiment, each student research team is required to hypothesize experimental outcomes from their designed immunoprecipitation. Do they expect to be able to immunoprecipitate their protein? Why or why not? What is the hypothesized molecular weight of their GFP-tagged protein? Do they expect to see any protein-protein interactions? If the immunoprecipitation does not work, how could the student research team improve upon their experimental design? Western blotting relies on the fact that proteins are separated by electrophoresis, transferred to a membrane, and then stained with antibodies to a specific target protein. All student research teams will use the same primary and secondary antibodies to detect the presence of GFP or GFP-tagged proteins. The day before, the instructor should add each student’s membrane to 5% milk in 1X PBS to block overnight. Students then prepare 10 mL of their rabbit anti-GFP primary antibody (Invitrogen #A11122; used at 1:1000 in 5% milk in 13 PBS) and allow their membranes to incubate for 30 min. After 30 min in primary antibody, students wash their blots for 5 min, three times each in 13 PBS 1 0.1% Tween-20 (Sigma #P2287). During the last wash, 10 mL of goat anti-rabbit secondary antibody conjugated to alkaline phosphatase is prepared (Thermo Scientific #31340; used at 1:5000 in 13 PBS). Membrane is incubated in secondary antibody for 30 min and then washed as described above. After washing, ensure that the membrane is protein side up (students will be able to tell based on the pre-stained ladder) and students add 2 mL of NBT/ BCIP solution (Thermo Scientific #34042) to cover the membrane. Students must visualize the blot as bands may appear very quickly or more gradually over a 15-min time period. Once bands are at the desired intensity, detection solution is rinsed off and water is added to stop the reaction. It is absolutely essential that students can interpret
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FIG 2
Student western blot of GFP-tagged Calmodulin immunoprecipitated from cleared Drosophila larval lysate. Purified GFP is used as a positive control to ensure that the Western blotting and immunoprecipitation with GFP-binding protein is working efficiently (lanes 1–4). The right-hand side of the gel demonstrates successful immunoprecipitation of GFP-tagged calmodulin from Drosophila larval lysate (lanes 6–10).
the results of their immunoblot staining and detection and determine if: 1) their western blot worked based on visualization of a band with a size corresponding to the size of their protein plus the size of GFP and 2) if their immunoprecipitation was successful (Fig. 2). Students need to take pictures of their immunoblots for their laboratory notebooks and for their up-coming presentations.
Week 10: Student Research Team Presentations Student research teams are responsible for researching the Drosophila GFP-tagged human disease homologue that they chose to immunoprecipitate from larval extracts. Each team creates and delivers an oral presentation with visual aids. Presentation guidelines explaining what information should be covered are provided to the students. Briefly, presentations begin with a general introduction and include methods used, predicted experimental outcomes, and obtained results from all experiments conducted throughout the semester. Additionally, student research teams are required to research their protein of choice and explore any interesting information based on recent scientific literature, include references, and draw conclusions from their experimental outcomes. Teams must also include the following crucial information about their particular protein: molecular weight, protein localization, possible interacting proteins, human homologs, mutant phenotypic information, and a detailed explanation of the protein’s normal cellular function and how this function is altered in the disease state.
Laboratory Course for Teaching Protein Purification
TABLE 2
Course evaluations demonstrate a high degree of student satisfaction with the course
Course evaluation
Strongly agree
Agree
Neutral
Disagree
Strongly disagree
N
I would recommend this course to other students
84.6%
15.4%
0%
0%
0%
26
How effective was this course in helping you learn, compared with other science courses you have had at your institute?
73.1
23.1
3.8
0
0
26
As a student, I understand the central concepts and ideas in this course
83.3
16.7
0
0
0
24
As a student, I can apply information/skill learned in this course
73.9
26.1
0
0
0
23
Overall, I would rate this course as a valuable learning experience
79.2
20.8
0
0
0
24
Course evaluations were compiled by IOTA solutions and presented as percentages. The data represent the combined results for courses taught in the fall semesters of 2012–2013.
Student Evaluation and Feedback All students were encouraged to complete an anonymous on-line course evaluation; 100% of students that participated in the voluntary evaluation reported an understanding of central concepts, the ability to apply the information and skills learned in the course, and agreed that the course overall was a valuable learning experience (Table 2). One student did not complete the evaluation, and one student left one question blank. The results indicate that 100% of students either “strongly agree” or “agree” that they would recommend this course to other students, that the course was effective in helping them learn compared with other science courses at Quinnipiac University, that they understood the central concepts and ideas, and that they can apply the information learned from course. In addition, the students all agreed that overall they would rate this course as a valuable learning experience (Table 2). Student evaluations and feedback indicate that the course was successful in providing students with practical laboratory skills and the theory behind each experiment performed. In particular, students enjoyed the on-line lectures and commented that they were useful in teaching the concepts of each experiment and thus prepared the students with the necessary knowledge to apply what they learned to the weekly experiments. Presenting core concepts outside of designated laboratory time allowed for more hands-on experimentation, which helped students, build confidence in their laboratory skills. Additionally, students commented that they enjoyed the built-in problemsolving that occurs during protein purification. The ability to make predictions and track proteins through a multistep protein purification was both an exciting and rewarding
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experience for students. Future development of this course will include presurvey and postsurvey assessment of research interest, practical laboratory skills, and knowledge of protein purification.
Conclusions A large number of laboratory courses have been developed which strive to meld the two worlds of teaching and research [2, 12–14]. Described here is a novel semesterlong laboratory course in protein purification that allows students hands-on experience in a wide-range of techniques while allowing instructors the versatility to incorporate their own research interests and goals. The material presented in these laboratory exercises give students the chance to hone their critical and quantitative reasoning skills while contributing to the scholarly research of the instructor. The course provides a solid background in techniques that students will utilize in additional laboratory courses, graduate or doctoral programs, or in the field of biotechnology. Importantly, the course can be modified to allow instructors to incorporate their research interests in vast areas of concentration due to the ubiquity of GFP in cell and molecular biology. Since GFP and GFP-tagged proteins are universal tools in biology, experiments can be continually adjusted to involve such techniques as chromatin immunoprecipitation and investigation of interaction proteins by mass spectroscopy [7].
Acknowledgments The authors are grateful to the contributions of Dr. Mary Matyskiela to manuscript editing and preparation. The
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Biochemistry and Molecular Biology Education authors would also like to thank Dr. Alex DeLencastre for helping prepare laboratory materials and joining in the adventure of teaching in a “flipped” classroom. A special thanks goes out to the graduate teacher assistants (Nick Vitale and Michael DiBiasio-White), and all MCB graduate students who participated in the Bio606 course at Quinnipiac University between 2012 and 2013.
References [1] Kuh, G. D., Chen, D., and Nelson Laird, T. F. (2007) Why teacherscholars matter: Some insights from FSSE and NSSE. Liberal Educ. 93, 40–45. [2] Fukami, T. (2013) Integrating inquiry-based teaching with faculty research. Science 339, 1536–1537. [3] Larkin, P. D. and Hartberg, Y. (2005) Development of a green fluorescent protein-based laboratory curriculum. Biochem. Mol. Biol. Educ. 33, 41–45. [4] Sommer, C. A., Silva, F. H., and Novo, M. T. M. (2004) Teaching molecular biology to undergraduate biology students. Biochem. Mol. Biol. Educ. 33, 7–10. [5] Giron, M. D. and Salto, R. (2011) From green to blue: Site-directing mutagenesis of the green fluorescent protein to teach protein structurefunction relationships. Biochem. Mol. Biol. Educ. 39, 309–315.
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[6] National Research Council. (2003) Bio2010: Transforming Undergraduate Education for Future Research Biologists, National Academies Press, Washington, DC. [7] Rothbauer, U., Zolghadr, K., Muyldermans, S., Schepers, A., Cardoso, M. C., and Leonhardt, H. (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol. Cell. Proteomics. 7, 282–289. [8] Siontorou, C. G. (2013) Nanobodies as novel agents for disease diagnosis and therapy. Int. J. Nanomed. 8, 4215–4227. [9] Morin, X., Daneman, R., Zavortink, M., and Chia, W. (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl. Acad. Sci. U S A 98, 15050– 15055. [10] Bollag, D. M., Rozycki, M. D., and Edelstein, S. J. (1996) Protein Methods, 2nd ed., Wiley-Liss, New York. [11] Elder, L. and Paul, R. (2005) The miniature guide to the art of asking essential questions. Foundation for Critical Thinking Press, USA. [12] Mitchell, B. F., and Graziano, M. R. (2006) From organelle to protein gel: A 6-wk laboratory project on flagellar proteins. CBE Life Sci. Educ. 5, 239–246. [13] Stahelin, R. V., Forslund, R. E., Wink, D. J., and Cho, W. (2003) Development of a biochemistry laboratory course with a project-oriented goal. Biochem. Mol. Biol. Educ. 31, 106–112. [14] Parrat, K. J., Osgood, M. P., Pappas, D. L., Jr. (2010) A research-based laboratory course designed to strengthen research-teaching nexus. Biochem. Mol. Biol. Educ. 38, 172–179.
Laboratory Course for Teaching Protein Purification
Christopher W. Carroll† Lani C. Keller‡*
From the †Department of Cell Biology, Yale University, New Haven, Connecticut 06520, ‡Department of Biological Sciences, Quinnipiac University, Hamden, Connecticut 06518
Abstract This adaptable graduate laboratory course on protein purification offers students the opportunity to explore a wide range of techniques while allowing the instructor the freedom to incorporate their own personal research interests. The course design involves two sequential purification schemes performed in a single semester. The first part comprises the expression and purification of a recombinant GFP-binding protein from E. coli. The student-purified GFPbinding protein is then used in the second part of the course to immunoprecipitate GFP-tagged proteins, and their potential interacting partners, from cell or tissue extracts. As an example, we describe the immunoprecipitation of GFP-tagged proteins from Drosophila melanogaster
larval extracts that are homologous to proteins implicated in human diseases, followed by western blotting to examine student experimental outcomes. However, the widespread availability of GFP-fusion proteins in diverse organisms enables researchers to tailor the second part of the course to their specific research programs while maintaining the flexibility to engage students in active learning. Student evaluations indicate a genuine excitement for research and in depth knowledge of both the techniques C 2014 by The performed and the theory behind them. V International Union of Biochemistry and Molecular Biology, 42(6):486–494, 2014.
Keywords: integration of research into undergraduate teaching; laboratory exercises; new course development; green fluorescent protein; molecular biology; Drosophila; protein purification
Introduction Teacher-scholars are committed to providing high-quality education while maintaining an active research program. The teacher-scholar model promotes the idea that students engaged in research gain a deeper understanding of material presented in traditional classroom settings, and have an enhanced ability to integrate and synthesize information [1]. For teaching and research to be mutually beneficial, it is essential to combine them synergistically [2]. Here, we outline a semester long laboratory course in protein purification that enables teacher-scholars to engage students in research-oriented, active learning while performing experi-
*Address for correspondence to: Department of Biological Sciences, Quinnipiac University, Hamden, Connecticut 06518, USA. E-mail: [email protected] Received 15 April 2014; Accepted 7 September 2014 DOI 10.1002/bmb.20827 Published online 21 October 2014 in Wiley Online Library (wileyonlinelibrary.com)
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ments that can be tailored to the research interests of the teacher-scholar. The use of green fluorescent protein (GFP) fusion proteins in laboratory classes is ideal for teacher-scholars. Educators have effectively employed GFP as a tool to teach the fundamental principles of protein purification in vitro, and it has been used to involve students in structurefunction studies through site-directed mutagenesis and spectrophotometry [3–5]. However, these approaches do not allow the instructor the flexibility to incorporate their own research interests into the course material. The course described here provides an opportunity for students to engage in research-oriented learning, as recommended by the National Research Council, while contributing to the research program of the instructor [6]. Moreover, the widespread use and availability of GFP-fusion proteins in virtually all academic research settings allows students and instructors alike a vast array of choices for material sources and biological problems to explore. Thus, students are empowered to take ownership of their projects while teacher-scholars are provided the flexibility to incorporate their own research interests into the course.
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The course is broken down into two integrated protein purification procedures. The first involves the expression and purification of a high affinity GFP-binding protein from bacteria. The GFP-binding protein consists of a fragment from a single-domain camelid antibody engineered to bind GFP with high affinity [7]. The sequence of the GFP-binding protein is cloned downstream of a 6-Histidine epitope tag in a bacterial expression vector, permitting purification by Nickel-affinity column chromatography using standard biochemical techniques. Single chain antibodies hold great promise as potential therapeutics and expose students to an emerging class of reagents that are increasingly important in academic and industrial research settings [8]. The subsequent procedure incorporates active learning as a method of exploration. The student-purified GFP-binding protein is coupled to a solid support and used to isolate a GFP-tagged fusion protein and its interacting proteins [3]. Described here is a procedure for the immunoprecipitation of GFP-fusion proteins from Drosophila larval extracts that are prepared from freely available reagents described in the GFP protein trap database (flytrap.med.yale.edu) [9]. Early in the semester, student research teams choose a Drosophila homolog of a human disease protein that they are interested in studying. They are then responsible for designing immunoprecipitation experiments to test hypotheses about protein localization, protein interaction partners, and ultimately the molecular mechanisms leading to disease. Student feedback demonstrated excitement about research, and a positive learning environment.
Course Synopsis The described course has been offered since 2012 and exists as an updated version of a long-running protein laboratory techniques course, which is one of five core courses required for all students in the Molecular and Cell Biology Master’s program at Quinnipiac University. Enrollment is usually between 30 and 45 students each fall, which are split into two or three sections with an average of 12–15 students per section. Acceptance into the Molecular and Cell Biology or Biomedical Science graduate program is required and graduate level biochemistry is a prerequisite. This laboratory class is taught in an interactive, hands-on learning format as a semester-long 4-hr/week introductory course. It could easily be adapted, however, to be delivered as an accompanying laboratory within upper-level undergraduate courses in biochemistry, molecular biology, or cell biology. Teams of two to three students are created on the first day of the course and act as collaborative student research teams throughout the semester. Each research team works in a technology pod with a computer providing continual access to the Internet, on-line lecture material, laboratory protocols, and all necessary programs and databases such as Excel, FlyBase (http://flybase.org/), and the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov/). These
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collaborative research teams serve to enhance learning by encouraging students to apply their knowledge, problemsolve and actively engage in concurrent data analysis. Lecture material was provided exclusively through on-line lectures, and students were expected to complete assigned reading from [10] and watch the on-line course material before class to allow for full participation in problem- solving and hands-on experimentation. The required text [10] provides an entirely self-contained practical and simple guide to protein purification. Although slightly out of date, this text is a tried and true reference that delivers easy to understand step-by-step procedures with clear illustrations for the majority of routinely used techniques in protein purification. This reference text was supplemented with current materials such as primary literature, manufacturer-supplied protocols, and in-depth explanations of contemporary topics such as nanobodies. On-line lecture material was assembled from a variety of these resources. A graduate assistant who previously took the course and an instructor who actively conducts research with Drosophila did all prelaboratory preparation. Stock solutions were made prior to each experiment to save time and allow for maximal student experimentation. Each research team was, however, required to prepare working solutions from provided stock solutions. Laboratory experiments were separated into two multipart sections with the goals of: 1) purifying a GFP-binding protein from E. coli and 2) using this GFP-binding protein to immunoprecipitate a GFP-tagged protein from Drosophila extracts (Table 1). Laboratory exercises were designed to incorporate a large range of protein purification techniques from cell lysis and affinity purification to immunoprecipitation and protein visualization by SDS-PAGE and immunoblotting. Importantly, while the first part of the course was standardized for all student research teams, each team selected a unique target protein for immunoprecipitation based on the research interests of the instructor and the students themselves in the second part of the course. Students were assessed through weekly in-class learning assessments, two laboratory exams based solely on the theory and practical applications of the material, class participation, and a laboratory notebook. In addition, each student research team prepared an oral presentation at the end of the course in which they provided the rationale for choosing a particular GFP-fusion protein for immunoprecipitation, candidate interacting proteins, and potential mechanisms by which mutations in the human homolog of the chosen protein could cause the associated human disease. The essential learning outcomes for the course included critical thinking and reasoning, scientific literacy, and quantitative reasoning.
Expenses Shared resources and supplies were crucial for designing and running this course, and all basic laboratory equipment
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TABLE 1
Week-by-week summary of laboratory experiments
Part I: GFP-Binding Protein Purification from bacteria 1
Bacterial cell lysis
2
Nickel affinity purification
3
Bradford protein assay
4
SDS-PAGE
5
Coupling GFP-Binding protein to beads
the instructor hoped the students would learn. It is important to define a set of essential questions that have the potential to motivate students to pursue facts and understand the basis of biological problems introduced within a course [11]. The essential questions that guided this course are: What makes the study of proteins more complicated and demanding than the study of nucleic acids? How have biotech companies developed methods to produce purified proteins like insulin? How could you discover all the proteins within a cell that interact with the Huntingtin protein, and why is this information important? How have advances in protein expression and purification impacted individuals and society?
Part II: Immunoprecipitation of GFP-tagged proteins 6
Lysis of samples expressing GFP-tagged protein
Laboratory Experiments
7
Immunoprecipitation of GFP-fusion proteins
Part I: GFP-Binding Protein Purification
8
SDS-PAGE
Prelaboratory Preparation of Bacterial Cells Expressing GFP-Binding Protein
9
Immunoblotting
10
Student Presentations
including a sonicator, a spectrophotometer, centrifuges, SDS-PAGE, and western blotting apparatuses were already on site. All chemicals and other reagents were either borrowed or purchased using funds specifically set aside for the Molecular and Cell Biology program within the Department of Biological Sciences at Quinnipiac University. Initial costs can be substantial, running anywhere between $500–2500; however, costs are considerably cheaper in subsequent course sessions because most reagents can be used for several semesters. Creativity and the simple action of reaching out to nearby institutes and professors goes a long way in providing the necessary reagents to fund a laboratory course like the one described here. The main costs associated with this course include desalting columns ($250), anti-GFP antibody and HRP-conjugated secondary antibody ($500), SimplyBlueTM SafeStain ($122.61), Ni-NTA agarose ($259- this can be used for many years for a variety of protein purification techniques), chemicals including imidazole, ampicillin, PMSF, NaCl, BME, Ponceau, and lysozyme ($375), NBT/ BCIP substrate solution ($124.44- can use for years), Bradford reagent ($127.00- can use for years), disposable columns ($210.00- enough to be used for many years), NHSActivated Agarose slurry ($210.00), and protein concentrators ($151.00- however this step can be easily substituted for dialysis which is significantly cheaper but requires treatment of samples in dialysis tubing with a high molecular weight osmotic like polyethylene glycol to initiate protein concentration).
Course Essential Questions Essential questions for the course were presented on the first day of class, within the syllabus and elaborated what
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BL21-CodonPlus (DE3)-RIPL cells (a bacterial strain carrying the gene for expression of the T7 polymerase and supplemented with extra copies of the arg, ile, leu, and pro tRNA genes; Fisher #NC9122855) were transformed with a pET28 6-HIS expression vector with the GFP-binding protein previously cloned in (developed for academic purposes only, available from corresponding author by request). Bacteria were plated on LB-agar plates (Sigma #L7025) supplemented with Kanamycin (Sigma #K1377). A single colony was chosen the next day and added directly to a 50 mL culture of 2X YT (2X Yeast Extract and Tryptone; Sigma #Y1003) media supplemented with 50 mg/mL of Kanamycin for overnight growth. This culture was then diluted back and grown in 2 L of 2X YT media at 37 C until an optical density of 0.8–1 was reached by spectrophotometric analysis at 600 nm. Expression of the GFP-binding protein was then induced with 0.1 mM IPTG (Sigma #I5502) and cultures were moved to 23 C. After growth for 20 hr, cells were then spun down, washed with 13 phosphate buffered saline (purchased as 103 PBS, Invitrogen #70011-044), pelleted in 50 mL conical tubes (BioExpress #C-3394-4), and stored at 270 C for future use.
Laboratory Experiment 1: Bacterial Cell Lysis During this laboratory, students learn the basics of cell lysis and use multiple methods to disrupt E. coli and liberate the highly expressed, soluble GFP-binding protein. Prior to beginning laboratory experiments, it is essential that all students are comfortable micropipetting. Instructors should therefore incorporate a pipetting activity the week prior to starting the purification scheme. Each student research team receives one aliquot of pelleted bacteria expressing the GFP-binding protein. Throughout the purification it is important to keep all samples on ice. Each research team is responsible for preparing 20 mL of lysis buffer [13 PBS with 300 mM NaCl
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(Sigma #S3014); 20 mM imidazole (Sigma #56750); 1 mM PMSF (Sigma #P7626 or Thermo Scientific #88660); 100 mg/mL lysozyme (Thermo Scientific #89833)] and gently resuspending their cell pellets. PMSF is a cytotoxic protease inhibitor and should only be handled with proper personal protection equipment such as gloves and laboratory goggles. Students then homogenize their sample by sonication on ice. Sonication produces dangerous high frequency sound waves so earplugs should be required while sonication is in progress. Each student research team then removes 100 mL of their homogenized sample and adds it to a microcentrifuge tube (BioExpress #C-3260-2) labeled “Whole Cell Lysate.” The remaining lysed bacterial extract is centrifuged at 10,000 3 g at 4 C for 20 min to generate a cleared lysate that contains the soluble GFP-binding protein. After centrifugation each student research team then removes 100 mL of supernatant and adds it to a microcentrifuge tube labeled “Cleared Lysate.” The remaining supernatant is saved for subsequent purification steps in a 50 mL tube labeled “Soluble Fraction.” The pellet can be discarded, although care should be taken to ensure that proper institutional recombinant DNA protocols are followed. At the end of this experiment, all student research teams should have three tubes stored at 220 C: (1) 20 mL Soluble Protein Fraction, (2) 100 mL Whole Cell Lysate, and (3) 100 mL Cleared Lysate.
Laboratory Experiment 2: Nickel-Affinity Purification of GFP-Binding Protein In this experiment, students acquire skills in affinity chromatography through a tried and true 6-HIS purification scheme. Each student research team starts this laboratory exercise by thawing their “Soluble Protein Fraction” and putting it on ice. Nickel affinity matrix (Ni-NTA, Qiagen #30210) is then prepared by pipetting 1.5 mL of 50% NiNTA slurry into a microcentrifuge tube and washing it three times in wash buffer (13 PBS 1 20 mM imidazole). The remaining “Soluble Protein Fraction” is then combined with the washed Nickel affinity resin and gently agitated at 4 C for 1 hr to allow binding. The cleared lysate and nickel affinity matrix mixture is then added to a plastic column (Thermo Scientific #29924) and 20 mL of flow through is collected and saved into a labeled 50 mL conical tube, of which 100 mL is transferred to a microcentrifuge tube labeled “Flow Through.” The chromatography column is washed three times with 5 mL of wash buffer to remove all non-specific binding of proteins to the affinity matrix. The 6-HIS-tagged GFP-binding protein is then eluted from the column by collecting ten 1 mL fractions with 13 PBS containing 500 mM imidazole. Note that high concentrations of imidazole can alter the pH of the solution; it is therefore critical to dilute the imidazole in the elution buffer from a 1 M stock that has been adjusted to pH 8.0. It is important for students to think critically about the process of affinity chromatography and to hypothesize where their 6-HIS-
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tagged GFP-binding protein will elute within the collected elution fractions. All elution fractions are labeled and stored at 220 C for further analysis.
Laboratory Experiment 3: Bradford Assay to Determine Protein Concentration of GFP-Binding Protein In this experiment, students learn how to create a standard curve demonstrating the relationship between absorbance at 595 nm and known concentrations of bovine serum albumin. Any available spectrophotometer capable of reading absorbance at 595 nm may be used for this exercise. Each student research team is responsible for creating their own standard curve within their technology pod, which is then used to extrapolate accurate protein concentrations of their GFP-binding protein. To create standard curves, students first must make a wide range of BSA concentrations (0, 125, 250, 500, 700, 1000, 1500, and 2000 mg/mL) from a 2.0 mg/mL stock solution (Sigma #P0834). Students are responsible for making all working solutions throughout the course to reinforce their quantitative reasoning skills. Once the standard curve BSA solutions are made, 10 mL of each is individually mixed with 500 mL of 13 Bradford reagent prepared according to manufacturer guidelines (Thermo Scientific #23236). The Bradford reagent, originally a red color, turns blue when bound to BSA, thus providing students with a means to qualitatively assess the protein concentration of their elution fractions prior to spectrophotometric analysis. The absorbance at 595 nm of each BSA solution is then determined by spectrophotometry and a standard curve (including line equation and R2 value to determine pipetting accuracy) is immediately created in Excel. Next 10 mL of each elution fraction is added to 500 mL of the Bradford reagent to determine the concentration of each elution fraction. Qualitative comparisons are made by eye before quantitatively measuring the absorbance of each protein fraction. The student-generated standard curves allow students to accurately determine the protein concentration of each of their protein fractions.
Laboratory Experiment 4: SDS-PAGE to Visualize Purification of GFP-Binding Protein In this exercise, students learn how to load, run, stain, and R analyze SDS-PAGE gels. Nu-PAGEV gels (Invitrogen #NP0321BOX) with MES SDS buffer (Invitrogen #NP0060) are utilized because full electrophoresis is complete within 30 minutes. Each student research team prepares the following samples by addition of sample buffer (Invitrogen #NP0007) supplemented with b-mercaptoethanol (Sigma #M3148) to break disulfide bonds: 20 mL “Whole Cell Lysate”, 20 mL “Cleared Lysate”, 20 mL column flowthrough, and 20 mL of elution fractions 1–6. bmercaptoethanol is hazardous and should only be used in the hood with proper personal protection equipment. Samples are then denatured by at 95 C for 10 min immediately before loading the gel. While the samples denature, Nu-
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Biochemistry and Molecular Biology Education R gels are prepared by rinsing them under water, PAGEV removing the tape and gel combs. Samples and 10 mL of prestained protein ladder (Invitrogen #10748-010), are then loaded and gels are run at 200 V for 30 min. It is important for students to ensure that there is a measurable current and to record in their laboratory notebooks what that current is. Students running gels that are not properly inserted into the apparatus may not show any current, while improper dilution of running buffer may cause the current to be higher than expected. Running the gels at a constant voltage of 200 V, should give an expected current of 110–125 mA at the start and 70–80 mA near the end. If students are unfamiliar with protein gel loading, it is important to have a practice gel and extra loading dye available. Gel-loading tips (Fisher #05–408-153) can also aid in the students’ ability to properly load protein gels. After electrophoresis, gels are carefully removed and stained with SimplyBlueTM Safestain (Invitrogen #LC6060) according to manufacturer guidelines. Students analyze their gels to investigate the purification of the soluble GFPbinding protein from cleared bacterial lysate (Fig. 1). Comparisons between student research team gels and between hypothesized results and obtained results are a great way to enhance critical reasoning skills. Results obtained by Bradford assay and by SDS-PAGE can also be compared to investigate purity of the samples. Each student research team must determine the three elution fractions with the highest concentration of purified GFP-binding protein and save these samples at 220 C for subsequent experimental steps.
Laboratory Experiment 5: Preparation of Purified GFPBinding Protein and Coupling to Agarose Beads The covalent coupling of purified and concentrated GFPbinding protein to a monovalent matrix, such as agarose beads, allows for subsequent fast and efficient one-step isolation of GFP fusion proteins from a sample of the instructor’s choosing. It is first important to remove the high concentrations of imidazole and to concentrate the GFPbinding protein to ensure efficient coupling. To provide students with additional column chromatography experience, size exclusion column chromatography is incorporated through the use of PD-10 desalting columns (Fisher #45000-148). Traditional dialysis may also be used to remove imidazole, although desalting columns provide a fast alternative. The advantages and disadvantages of choosing either dialysis or size exclusion chromatography should be discussed. To conduct desalting by size exclusion chromatography, each student research team must equilibrate the PD-10 column with equilibration buffer (13 PBS). The top three elution fractions from the previous laboratory exercise with the most protein according to Bradford and SDSPAGE are then combined and added to the column and flow-through is discarded. The soluble, desalted GFPbinding protein is then eluted and collected by the addition
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FIG 1
Student SDS-PAGE gel stained with SimplyBlueTM Safestain to visualize nickel-affinity purification of GFP-binding protein from E. coli. Whole cell lysate and the cleared lysate show large numbers of proteins expressed in E. coli with a band at 19 kD representing the highly expressed GFP-binding protein. Depletion of the GFP-binding protein in the column flow through indicates successful binding of the 6-HIS GFPbinding protein to the Nickel affinity chromatography column. Elutions 1–5 demonstrate purification of the GFP-binding protein with the highest expression in elution one and a tapering off there after. Arrow indicates successful purification of 6HIS-tagged GFP-binding protein.
of 3.5 mL 13 PBS. At this point the imidazole is not yet eluted and therefore remains within the matrix of the column. Following size exclusion chromatography, a 20 mL aliquot of the desalted GFP-binding protein is saved while the remaining protein is concentrated by centrifugation in Pierce Protein Concentrators with a 9 K Molecular Weight Cut Off (Thermo Scientific #89884A). A quick qualitative Bradford assay is conducted to determine if the GFPbinding protein concentration is in fact increased (sample after concentration should have a higher concentration of GFP-binding protein and therefore will be more blue than prior to centrifugation). Student research team then takes their remaining concentrated sample and covalently couples it to NHS-activated agarose beads by incubation for 20 min at room temperature with gentle rocking. Beads are prewashed with water three times prior to student use. To stop the coupling reaction, 500 mL of 100 mM Tris, pH 7.5 is added for 15 min and then removed by centrifugation. A quick Bradford assay can again be used to ensure that the purified GFP-binding protein is coupled to the beads by comparing a sample before coupling to the supernatant after coupling (supernatant after coupling should be less blue than starting sample because the purified GFPbinding protein is now coupled to the agarose beads).
Laboratory Course for Teaching Protein Purification
Reintroducing the Bradford assay multiple times throughout the course reinforces the importance of following a protein through a protein purification procedure and allows students to make hypotheses at many points throughout the protocol. The coupled agarose beads are stored at 4 C and are washed once in 13 PBS prior to being used in immunoprecipitation experiments.
Part II: Immunoprecipitation from Drosophila The second half of the course allows students to take more ownership of their project and engage in active and collaborative learning within their research teams. Teach student research team selects a human disease of their interest to research. They are required to determine what genes and proteins are disrupted or involved in causing the disease state and establish which genes have homologues in Drosophila. The student research teams use FlyBase (http://flybase.org/), the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), and FlyTrap (http:// flytrap.med.yale.edu/) databases to learn about their genes/ proteins, find publicly available GFP-tagged lines, and make predictions about protein localizations, expression levels, and protein-protein interactions. Student research teams then design and perform immunoprecipitation experiments using their purified GFP-binding protein from the first half of the semester. The basic immunoprecipitation procedure was similar between research teams and is described in detail below, however the experimental details and hypotheses that each research team asked were unique. This process promotes active learning, reading, writing, discussion, and problem-solving skills and culminated in the oral presentation of their work to the class. The presentations were required to include their experimental procedures and results, descriptions of the known molecular mechanisms causing the disease in humans, and explanations of how the use of model organisms may help in understanding and potential treatment of their particular disease.
Laboratory Experiment 6: Lysis of Drosophila Larva Expressing GFP-tagged Proteins The second part of this laboratory course focuses on the isolation of GFP fusion proteins from the model organism Drosophila, although it is important to note that instructors may employ any GFP-expressing model organism or in vitro system that is relevant to their research interests. In the laboratory exercise described here, students attain hands-on experience working with Drosophila and compare and contrast cell lysis procedures used previously for bacteria to those used for Drosophila larva. The instructor must preorder Drosophila lines expressing GFP-tagged proteins based on student research team interest from FlyTrap (http://flytrap.med.yale.edu/) and ensure that each team has 50 Drosophila larva or adults depending on their experimental design. The GFP-expressing larva are collected and frozen at 280 C for 10 min to increase lysis efficiency.
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Larva are then thawed and exposed to 200 mL of fly lysis buffer (1X PBS; 150 mM EDTA; 0.1% NP-40; 10% glycerol; 1 mM PMSF) before homogenization in an microcentrifuge tube with a Kontes motorized tissue grinder (Fisher #K749540-0000) and RNase-Free disposable pellet pestles (Fisher #12–141-364). Homogenized Drosophila larva expressing GFP fusion proteins are then centrifuged at high speed for 15 min at 4 C to pellet nonsoluble proteins. The supernatant containing all soluble proteins are saved and stored at 220 C for the following week.
Laboratory Experiment 7: Immunoprecipitation of GFP Fusion Proteins Immunoprecipitation is a technique for isolating a protein from a complex mixture using an antibody that specifically binds that particular protein. In this laboratory, the GFPbinding protein (nanobody acting as an antibody) is coupled to NHS-activated agarose beads (Thermo Scientific #26200) to precipitate the GFP fusion protein (antigen) from Drosophila lysate. Purified GFP can act as a positive control to ensure that coupled beads have the capacity to precipitate GFP, if instructors have access to it. Each student research team first thaws their cleared Drosophila lysate and removes a 10 mL aliquot, which is labeled “input.” The “input” is mixed directly with sample buffer and stored at 220 C. The cleared Drosophila lysate is then added to the agarose beads coupled to GFP-binding protein and incubated at 4 C for 1 hr with gentle rocking. The 1-hr incubation period allows for class discussion of immunoprecipitation procedure and predictions of expected outcomes. The GFP-binding protein on the agarose beads should bind with high affinity to any GFP-tagged protein within the Drosophila larval lysate. After the 1 hr binding step, agarose beads are centrifuged down and a 10 mL aliquot of the supernatant is removed and labeled “supernatant.” Sample buffer is added directly to the “supernatant” and stored at 220 C. Samples are then recentrifuged to pellet agarose-beads and washed three times with 13 PBS. During washes, care must be taken to avoid pipetting beads, which will be bound to GFP-tagged proteins and any interacting proteins. Pelleted beads are then resuspended directly in 20 mL of sample buffer and stored at 220 C.
Laboratory Experiment 8: SDS-PAGE and Transfer onto Western Blot for Immunoblotting Each student research team prepares the following samples for SDS-PAGE: 1) prestained protein ladder, 2) purified GFP “input” as a positive control, 3) “supernatant” after immunoprecipitation of purified GFP, 4) agarose beads after immunoprecipitation with purified GFP, 5) prestained protein ladder, 6) Drosophila cleared lysate “input”, 7) “supernatant” after immunoprecipitation, and 8) agarose beads after immunoprecipitation with GFP-tagged protein R gel bound. Samples are then loaded and in a Nu-PAGEV and exposed to 200 V for 30 min as described above. Once again, it is important for students to ensure that there
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Biochemistry and Molecular Biology Education is a measurable current and to record in their laboratory notebooks what that current is. After electrophoresis, gels are carefully removed and traditional western blot transfer R is set up using NuPAGEV transfer buffer (Invitrogen #NP0006-1) and pre-prepared nitrocellulose filter paper sandwiches, 0.45-mm pore size (Invitrogen #LC2006). The use of a prestained ladder allows for students to visualize effective transfer onto nitrocellulose membrane. To investigate total protein transferred onto membrane, students perform Ponceau (Sigma #P3504) staining of the nitrocellulose membrane. Students should make predictions about what proteins bind to the nitrocellulose versus those proteins that will be visible after western blot analysis. After Ponceau staining, student observations are made and nitrocellulose membrane is washed with distilled water, dried, and stored at room temperature.
Laboratory Experiment 9: Immunoblotting and Detection of Immunoprecipitated GFP-tagged Protein from Drosophila Prior to starting this laboratory experiment, each student research team is required to hypothesize experimental outcomes from their designed immunoprecipitation. Do they expect to be able to immunoprecipitate their protein? Why or why not? What is the hypothesized molecular weight of their GFP-tagged protein? Do they expect to see any protein-protein interactions? If the immunoprecipitation does not work, how could the student research team improve upon their experimental design? Western blotting relies on the fact that proteins are separated by electrophoresis, transferred to a membrane, and then stained with antibodies to a specific target protein. All student research teams will use the same primary and secondary antibodies to detect the presence of GFP or GFP-tagged proteins. The day before, the instructor should add each student’s membrane to 5% milk in 1X PBS to block overnight. Students then prepare 10 mL of their rabbit anti-GFP primary antibody (Invitrogen #A11122; used at 1:1000 in 5% milk in 13 PBS) and allow their membranes to incubate for 30 min. After 30 min in primary antibody, students wash their blots for 5 min, three times each in 13 PBS 1 0.1% Tween-20 (Sigma #P2287). During the last wash, 10 mL of goat anti-rabbit secondary antibody conjugated to alkaline phosphatase is prepared (Thermo Scientific #31340; used at 1:5000 in 13 PBS). Membrane is incubated in secondary antibody for 30 min and then washed as described above. After washing, ensure that the membrane is protein side up (students will be able to tell based on the pre-stained ladder) and students add 2 mL of NBT/ BCIP solution (Thermo Scientific #34042) to cover the membrane. Students must visualize the blot as bands may appear very quickly or more gradually over a 15-min time period. Once bands are at the desired intensity, detection solution is rinsed off and water is added to stop the reaction. It is absolutely essential that students can interpret
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FIG 2
Student western blot of GFP-tagged Calmodulin immunoprecipitated from cleared Drosophila larval lysate. Purified GFP is used as a positive control to ensure that the Western blotting and immunoprecipitation with GFP-binding protein is working efficiently (lanes 1–4). The right-hand side of the gel demonstrates successful immunoprecipitation of GFP-tagged calmodulin from Drosophila larval lysate (lanes 6–10).
the results of their immunoblot staining and detection and determine if: 1) their western blot worked based on visualization of a band with a size corresponding to the size of their protein plus the size of GFP and 2) if their immunoprecipitation was successful (Fig. 2). Students need to take pictures of their immunoblots for their laboratory notebooks and for their up-coming presentations.
Week 10: Student Research Team Presentations Student research teams are responsible for researching the Drosophila GFP-tagged human disease homologue that they chose to immunoprecipitate from larval extracts. Each team creates and delivers an oral presentation with visual aids. Presentation guidelines explaining what information should be covered are provided to the students. Briefly, presentations begin with a general introduction and include methods used, predicted experimental outcomes, and obtained results from all experiments conducted throughout the semester. Additionally, student research teams are required to research their protein of choice and explore any interesting information based on recent scientific literature, include references, and draw conclusions from their experimental outcomes. Teams must also include the following crucial information about their particular protein: molecular weight, protein localization, possible interacting proteins, human homologs, mutant phenotypic information, and a detailed explanation of the protein’s normal cellular function and how this function is altered in the disease state.
Laboratory Course for Teaching Protein Purification
TABLE 2
Course evaluations demonstrate a high degree of student satisfaction with the course
Course evaluation
Strongly agree
Agree
Neutral
Disagree
Strongly disagree
N
I would recommend this course to other students
84.6%
15.4%
0%
0%
0%
26
How effective was this course in helping you learn, compared with other science courses you have had at your institute?
73.1
23.1
3.8
0
0
26
As a student, I understand the central concepts and ideas in this course
83.3
16.7
0
0
0
24
As a student, I can apply information/skill learned in this course
73.9
26.1
0
0
0
23
Overall, I would rate this course as a valuable learning experience
79.2
20.8
0
0
0
24
Course evaluations were compiled by IOTA solutions and presented as percentages. The data represent the combined results for courses taught in the fall semesters of 2012–2013.
Student Evaluation and Feedback All students were encouraged to complete an anonymous on-line course evaluation; 100% of students that participated in the voluntary evaluation reported an understanding of central concepts, the ability to apply the information and skills learned in the course, and agreed that the course overall was a valuable learning experience (Table 2). One student did not complete the evaluation, and one student left one question blank. The results indicate that 100% of students either “strongly agree” or “agree” that they would recommend this course to other students, that the course was effective in helping them learn compared with other science courses at Quinnipiac University, that they understood the central concepts and ideas, and that they can apply the information learned from course. In addition, the students all agreed that overall they would rate this course as a valuable learning experience (Table 2). Student evaluations and feedback indicate that the course was successful in providing students with practical laboratory skills and the theory behind each experiment performed. In particular, students enjoyed the on-line lectures and commented that they were useful in teaching the concepts of each experiment and thus prepared the students with the necessary knowledge to apply what they learned to the weekly experiments. Presenting core concepts outside of designated laboratory time allowed for more hands-on experimentation, which helped students, build confidence in their laboratory skills. Additionally, students commented that they enjoyed the built-in problemsolving that occurs during protein purification. The ability to make predictions and track proteins through a multistep protein purification was both an exciting and rewarding
Carroll and Keller
experience for students. Future development of this course will include presurvey and postsurvey assessment of research interest, practical laboratory skills, and knowledge of protein purification.
Conclusions A large number of laboratory courses have been developed which strive to meld the two worlds of teaching and research [2, 12–14]. Described here is a novel semesterlong laboratory course in protein purification that allows students hands-on experience in a wide-range of techniques while allowing instructors the versatility to incorporate their own research interests and goals. The material presented in these laboratory exercises give students the chance to hone their critical and quantitative reasoning skills while contributing to the scholarly research of the instructor. The course provides a solid background in techniques that students will utilize in additional laboratory courses, graduate or doctoral programs, or in the field of biotechnology. Importantly, the course can be modified to allow instructors to incorporate their research interests in vast areas of concentration due to the ubiquity of GFP in cell and molecular biology. Since GFP and GFP-tagged proteins are universal tools in biology, experiments can be continually adjusted to involve such techniques as chromatin immunoprecipitation and investigation of interaction proteins by mass spectroscopy [7].
Acknowledgments The authors are grateful to the contributions of Dr. Mary Matyskiela to manuscript editing and preparation. The
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Biochemistry and Molecular Biology Education authors would also like to thank Dr. Alex DeLencastre for helping prepare laboratory materials and joining in the adventure of teaching in a “flipped” classroom. A special thanks goes out to the graduate teacher assistants (Nick Vitale and Michael DiBiasio-White), and all MCB graduate students who participated in the Bio606 course at Quinnipiac University between 2012 and 2013.
References [1] Kuh, G. D., Chen, D., and Nelson Laird, T. F. (2007) Why teacherscholars matter: Some insights from FSSE and NSSE. Liberal Educ. 93, 40–45. [2] Fukami, T. (2013) Integrating inquiry-based teaching with faculty research. Science 339, 1536–1537. [3] Larkin, P. D. and Hartberg, Y. (2005) Development of a green fluorescent protein-based laboratory curriculum. Biochem. Mol. Biol. Educ. 33, 41–45. [4] Sommer, C. A., Silva, F. H., and Novo, M. T. M. (2004) Teaching molecular biology to undergraduate biology students. Biochem. Mol. Biol. Educ. 33, 7–10. [5] Giron, M. D. and Salto, R. (2011) From green to blue: Site-directing mutagenesis of the green fluorescent protein to teach protein structurefunction relationships. Biochem. Mol. Biol. Educ. 39, 309–315.
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[6] National Research Council. (2003) Bio2010: Transforming Undergraduate Education for Future Research Biologists, National Academies Press, Washington, DC. [7] Rothbauer, U., Zolghadr, K., Muyldermans, S., Schepers, A., Cardoso, M. C., and Leonhardt, H. (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol. Cell. Proteomics. 7, 282–289. [8] Siontorou, C. G. (2013) Nanobodies as novel agents for disease diagnosis and therapy. Int. J. Nanomed. 8, 4215–4227. [9] Morin, X., Daneman, R., Zavortink, M., and Chia, W. (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl. Acad. Sci. U S A 98, 15050– 15055. [10] Bollag, D. M., Rozycki, M. D., and Edelstein, S. J. (1996) Protein Methods, 2nd ed., Wiley-Liss, New York. [11] Elder, L. and Paul, R. (2005) The miniature guide to the art of asking essential questions. Foundation for Critical Thinking Press, USA. [12] Mitchell, B. F., and Graziano, M. R. (2006) From organelle to protein gel: A 6-wk laboratory project on flagellar proteins. CBE Life Sci. Educ. 5, 239–246. [13] Stahelin, R. V., Forslund, R. E., Wink, D. J., and Cho, W. (2003) Development of a biochemistry laboratory course with a project-oriented goal. Biochem. Mol. Biol. Educ. 31, 106–112. [14] Parrat, K. J., Osgood, M. P., Pappas, D. L., Jr. (2010) A research-based laboratory course designed to strengthen research-teaching nexus. Biochem. Mol. Biol. Educ. 38, 172–179.
Laboratory Course for Teaching Protein Purification
