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Keywords:

  • Protein purification;
  • His tagging;
  • GST fusion proteins

Abstract

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. Acknowledgements
  7. REFERENCES

Two distinct courses, “Molecular Biology” taught by the Biology Department and “Advanced Biochemistry” taught by the Chemistry Department, complement each other and, when taught in a coordinated and integrated way, can enhance student learning and understanding of complex material. “Molecular Biology” is a comprehensive lecture-based course with a 3-h laboratory once a week, while “Advanced Biochemistry” is a completely laboratory-based course with lecture fully integrated around independent student projects. Both courses emphasize and utilize cutting-edge technology. Teaching across departmental boundaries allows students access to faculty expertise and techniques rarely used at the undergraduate level, namely the tagging of proteins and their use in protein purification.

With the evolution of Molecular Biology as a separate discipline from Biochemistry, educators have had to devise better pedagogy to illustrate both the overlap and the uniqueness of each field, especially in the laboratory [1, 2]. This article illustrates an attractive approach to this dilemma by offering both “Molecular Biology” and an “Advanced Biochemistry” course that are separate but complementary. At Simmons College, as at most colleges, students generally complete “Molecular Biology” and “Advanced Biochemistry” courses in their junior or senior year. These one-semester courses are taught in the same semester, with “Advanced Biochemistry” offered every year and “Molecular Biology” every other year. At this point, students have had one-semester courses in “Cell Biology,” “Genetics,” and “Biochemistry.” The majority of the students take both courses either sequentially or concurrently, which is possible because these courses complement each other. With the “Molecular Biology” course offered every other year, the number of students is about twice that of “Advanced Biochemistry.” Two laboratory sections are offered with “Molecular Biology” so that each student can learn the techniques and feel that she is contributing to the laboratory project. The smaller size of “Advanced Biochemistry,” averaging about 12 students each year, is an advantage and is crucial to maintaining the independence of the students and the student-driven laboratories as described below. This article reviews a single semester's experience when both courses were taught simultaneously.

In “Molecular Biology,” the students learned what is involved in cloning a gene and, in the process, generated a tagged protein. The students in “Advanced Biochemistry” started with a tagged protein and then designed strategies to purify the protein. Both glutathione S-transferase (GST)11 (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and His (Qiagen, Inc., Valencia, CA) tags were used, and students researched the advantages and disadvantages of each type of tag.

“Molecular Biology” is a comprehensive course, which incorporated an intensive lecture twice a week with a detailed, experimentally based text, and one 3-h laboratory per week. The most recent text used for this one-semester course was Molecular Biology by Robert F. Weaver (McGraw-Hill Co.) The key focus of both the course and the text was mechanisms of how genes are replicated, maintained, and expressed in cells and covered both prokaryotes (bacteria and phage) and eukaryotes. The transcriptional machinery of prokaryotes and eukaryotes was examined in great detail and how it is regulated by transcription factors for activation and repression. The effects of chromatin structure on transcription were included as well along with histone modification and chromatin remodeling protein complexes. Eukaryotic modifications of RNA were studied, which included capping, splicing, and polyadenylation of mRNA followed by translation and its regulation by RNA interference. Other topics included DNA replication, homologous and site-specific recombination, transposition, and the course ended with genomic and proteomic analyses. The lectures and laboratories were closely linked and were enhanced by a cloning project to tag and overexpress a gene of interest.

“Advanced Biochemistry” was designed to give students the opportunity to study advanced topics and methodology through laboratory experience. It has been demonstrated that experience with real-life research problems enhances student learning of advanced topics [3]. “Advanced Biochemistry” was presented as a research-based course open to students of the Colleges of the Fenway, which includes not only Simmons College, but also Massachusetts College of Pharmacy, Emmanuel College, Wheelock College, and Wentworth Institute of Technology. Students underwent a graduate-type laboratory experience as they purified two GST-tagged proteins. As they were performing experiments, they were addressing advanced topics by way of designing, implementing, troubleshooting, and analyzing real-world biochemistry. They had to research the theory behind the technique in order to troubleshoot what wasn't working and understand why it worked under other circumstances. They used current scientific literature, the Internet, and company-provided protocols to review the background material and current methodologies. There was no lecture, but rather many short one-on-one conversations with the instructor, other students, or other faculty. When important theory arose during one of these discussions, the instructor gave a general lecture to the whole group so that all had access to topics raised. Take-home quizzes also provided a means for the students to explore common topics in greater depth, while still preserving the independent nature of this course. Table I summarizes the approaches taken in both “Advanced Biochemistry” and “Molecular Biology.”

RESULTS

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. Acknowledgements
  7. REFERENCES

“Molecular Biology”—

In class, while the overall format was lecture-based, the students actively participated by examining figures and gels from the text. The lecture outline was available before class on the course website so students could familiarize themselves with the upcoming lecture. This meant more class time was available to analyze experimental data and to discuss articles, which were also available on-line. Techniques used in experiments were described and tied to techniques being used in the laboratory section of “Molecular Biology.” Approaches to research were also discussed in the context of the 50th anniversary of the discovery of the structure of DNA and the difference between discovery-based and hypothesis-driven research. Students were encouraged to participate and comments made by the group were written on the board for later reference. Some of these same issues appeared on quizzes or exams (see assessment section).

The students in this “Molecular Biology” course successfully cloned two genes from the bacterial chromosome, the hns gene (described below) and a gene of unknown function, yjjQ. The hns gene encodes a DNA binding protein of ∼15,391 Da that acts pleiotropically in Escherichia coli as a global regulator of gene expression. The students cloned PCR-generated DNA carrying the chromosomal hns gene into a protein expression vector, verified the presence of the insert, and then used SDS-PAGE to visualize the protein. This cloning project, which started fairly early in the semester, gave the students a sense of ownership and involvement in the “Molecular Biology” laboratory section.

Qiagen generously donated the protein expression vector pQE31, which is designed both for inducible gene expression and the addition of a histidine (His6) tag on the N terminus of the expressed protein. Three vectors are available, one of which corresponds to the reading frame of the protein of interest. The sequence of the H-NS coding region was in a different reading frame than pQE31, and its gene needed to be adapted before cloning into pQE31. Students were given the assignment to design primers containing restriction enzyme sites and the proper number of nucleotides so that the amplified hns gene could be inserted into the vector with the hns sequence in frame with the his codons. This was a challenging assignment, which required that the students understand the process of amplification and how new restriction sites can be incorporated into a genetic sequence. They had to grapple with reading frames so that the His6 tag could be added to the H-NS protein. Suddenly reading frames, with which they have all been familiar, took on new meaning and became more than just a homework assignment. The students needed to figure this out in order to proceed in cloning the hns gene. Not only did they want to clone the gene, but also they wanted a properly translated protein.

The students were given microgram amounts of PCR products to facilitate cloning or set up the PCR themselves. The key point was they needed adequate amounts of PCR product to be successful in cloning. Qiagen kits were used for PCR purification, gel extraction, and minipreps (see below). The students digested the purified PCR product and the plasmid vector with restriction enzymes, purified the fragments by gel electrophoresis, and carried out a ligation reaction based on their estimates of vector to insert ratios. The next laboratory period, they transformed XL-1 Blue (Stratagene, La Jolla, CA), which is an excellent cloning strain. Nearly all of the students had transformants the next day, and in the following laboratory period, they isolated plasmid DNA from their transformants. To determine if they had actually cloned the hns gene, they digested their plasmid DNA with restriction enzymes and looked for the presence of an insert on an agarose gel. Three groups out of seven had plasmids containing the hns insert. E. coli strain BL21 (Pharmacia), which is useful for protein expression, was then transformed with the plasmids carrying the hns gene. Cultures of BL21 were grown with or without isopropyl β-D-thiogalactoside (IPTG) to induce expression of the gene. The students lysed the cells by boiling in SDS sample buffer and then carried out SDS-PAGE. The gel was stained with Coomassie Brilliant Blue R, and the H-NS protein could be clearly seen in the total protein extract in cultures induced with IPTG. The students succeeded in cloning a gene from the chromosome and visualized its protein product under conditions that they controlled.

Earlier in the laboratory sequence, the students learned how to isolate plasmid DNA, digest DNA, and transform cells. Now they used these methods with their own clones to determine whether or not their plasmids contained inserts. The repetition of these methods was beneficial in that they could see the utility of these techniques in cloning a gene. There was interest to see who had the insert as well. At each step of the cloning procedures, students realized that, for example, ligations may or may not work and transformation with the ligation mix may or may not result in transformants. They had to figure out what affects these procedures and try to come up with optimal conditions. Understanding induction became crucial as well; no longer was regulation of the lac operon an abstraction because the plasmid they were using had a phage T5 promoter regulated by two lac operator sequences.

“Advanced Biochemistry”—

The key strategy in this course was to have the students work independently without daily procedures being provided to them. There is a theme to the course each semester, with protein purification as the theme of the semester being discussed, and a suggested global methodology, such as using GST fusion purification techniques. To cut down on the reagents/resources that the instructor needed to provide, the students were given a defined range of methods. They formulated a hypothesis about an undetermined outcome and then designed and planned the experiments that would address that hypothesis. The students chose techniques within the loosely defined theme, implemented the experiments, and then reported to the instructor as an advisor. The students did all the background research themselves and used other students as resources. Then they chose the variables with which to experiment when trying to optimize methodology. There were weekly laboratory meetings where students received peer feedback on their experiments, just as in a graduate laboratory. They were required to maintain a detailed notebook and protocols book. The students approached the project with the expectation that a thesis in the form of a manuscript would be the final outcome.

As described above, students used basic biochemical and molecular biological techniques to purify a protein using the GST fusion protein purification system and followed suggested protocols provided by the manufacturer, Amersham Pharmacia Biotech, Inc. They were to purify and solubilize a protein to the extent that others could then use the pure protein in biological assays. They were given a choice of two proteins to work with: BglJ, a protein involved in anti-silencing in Eschericia coli, or FHOS, a novel formin protein with a potential role in signal transduction. These proteins were chosen because they are components of ongoing research in faculty laboratories here at Simmons. The use of real research problems enhanced the students' feelings of investment in the final outcome of their endeavors. Students were provided with background literature on the methodology and on the target proteins, as well as the manual that accompanies purchase of the materials from Pharmacia, which were graciously provided by the company [4]. At the start of the semester, the students were guided through the Pharmacia manual and they were given instruction to initiate the experiments. After the first 2 weeks, they were responsible for planning each subsequent experiment based on analysis of the data from the previous ones.

Initially, students needed to check the DNA that they were given to confirm that the plasmids did indeed contain the DNA sequences corresponding to bglJ or FHOS, respectively, which encoded the two proteins of interest. They isolated plasmid DNA using Qiagen kit technology and then they used restriction analysis to verify the presence of an insert of the expected size. They transformed the plasmid DNA containing the sequences encoding the GST protein fusions into BL21 cells. Then they began their quest for purification. The two proteins chosen for purification have markedly different structures and functions. This demonstrated key principles of how protein structure can affect solubility and whether or not a protein can be easily purified.

Upon verification of the cloned GST constructs, the students began their purification procedures, asking the relevant questions in order to determine the best strategies for experimentation. For example, students asked:

  • After transforming BL21 cells with the GST-BglJ plasmid and inducing expression of the fusion protein with IPTG at 37 °C, was a protein at the expected size observed on a Coomassie-stained SDS gel?

  • Was there expression of the GST protein alone as a control?

  • What was the length of induction time?

  • What growth temperature worked best for protein expression?

  • Did temperature affect potential solubility?

Each of these questions gave students opportunities to troubleshoot and enhance the protocols in order to optimize results. At this stage, the instructor was primarily available for consultation and guidance, but the ultimate decisions about what variables to manipulate were determined by the students. This gave the students a sense of ownership of the outcome and allowed them to feel fully involved in their research. As the students progressed, each group of students was ultimately pursuing different hypotheses and no two groups were performing the same experiment. This provided an environment similar to a real research laboratory and fostered communication between laboratory groups. Students used each other as sounding boards and colleagues, often sharing results that could help other groups in planning the next steps.

Students repeated the expression assays many times and manipulated some new aspect of the protocol to both enhance the amount of fusion protein expressed and to increase solubility. The goal was to produce the protein in sufficient quantities so that the GST tag could be cleaved and the purified protein used in enzymatic assays. They became experts with SDS-PAGE gels and Coomassie staining. They manipulated culture temperatures, culture media, protein concentrations, and induction times with each subsequent experiment. At the conclusion of this course, students were able to show successful expression of both GST-BglJ and GST-FHOS in BL21 cells. They subsequently demonstrated increased solubility of both fusion proteins upon manipulation of the induction time and culture temperature. They successfully performed a Western blot using a GST antibody probe and went on to cleave the GST-BglJ fusion using the thrombin enzyme to produce purified BglJ protein.

Active participation in the decision-making process contributed greatly to their comprehension of the difficulties facing a researcher at the bench. In “Advanced Biochemistry,” each group of students working on a particular project prepares a manuscript of their results. Like any laboratory setting, some projects are more feasible than others and some students make more progress. Because each group of students was responsible for a particular project, their manuscript reflected their progress. If the manuscript is of publishable quality, students were encouraged to submit it to a journal publishing student research with the assistance of the faculty members. Seeing laboratory work go from the bench to a manuscript is a powerful experience for all the students whether they are able to publish or not. Students felt a tremendous sense of accomplishment at the end of the semester, and one group went on to publish their results in the Journal of Undergraduate Chemistry Research [5].

DISCUSSION

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. Acknowledgements
  7. REFERENCES

For both courses, the instructors chose a theme that was based on ongoing research being performed at Simmons College so that students felt invested in the outcome of their work. The cloning project in “Molecular Biology” showed the students how research can lead to a useful product, namely a cloned and tagged gene, which can be used in further research projects. In “Advanced Biochemistry,” the knowledge that this was a “real” project that could ultimately result in a “real” published article was very important to them. They felt a responsibility in doing good, controlled work and, thus, were more committed. This was more than performing “canned” laboratories, just to learn techniques. It was research designed to answer legitimate scientific questions. However, because the process of cloning a particular gene to generate a tagged protein and then purifying that tagged protein can be applied to virtually any gene, these approaches can be used by instructors anywhere to involve their students in exciting research projects.

The advantages to the unique approach to the laboratory experience that the “Advanced Biochemistry” course offers are multi-faceted. Students learned by doing and by being engaged directly with the material. These were not preplanned experiences with known right or wrong answers, which generated anticipation and excitement for the students and gave them a real sense of what they will experience in an actual research laboratory. Student progress was measured not by “percent error” but by development and acquisition of trouble-shooting skills, commitment, and dedication to a final group goal. The initial questions were open-ended and each set of results led to a new set of possible experiments, which demonstrated the natural progress of real scientific research. Seeking scientific insights rarely follows a direct path but more frequently a series of possible side-roads. This course emphasized the creativity of real science and allowed students to think imaginatively to design a variety of experiments.

Assessment—

To assess how well the students in “Molecular Biology” learned the course material, a variety of graded activities were used in addition to traditional quizzes and exams and experimental write-ups. For example, given the research orientation of the course, a paper in the format of a review to an editor was assigned, which meant the students had to read and assess current research articles. The students learned how to analyze and interpret gels to see the results of an experiment. They weren't just reading text and memorizing facts. Actual data, including gels, were included in the quizzes and the exams and the students analyzed the data and wrote short explanatory essays. This experience helped them in their reviews of a research article where they had to decide as reviewers if they would have published the work. They had to make suggestions, back them up with experiments, and submit their recommendations as if they were writing a review to an editor of a journal. It was a demanding exercise that the students hadn't encountered in other classes. Also, on exams, they had to respond to written passages and write short explanations of their own. Finally, they wrote extensively in the laboratory and learned how to write more precisely.

For assessment of student knowledge, “Advanced Biochemistry” did not use an exam-based system but rather a written and oral interpretation method for assessment. There were weekly written summaries of current scientific literature designed to both enhance the students' knowledge of advanced topics and make them comfortable with reading and interpreting complex scientific articles. Initially, students were provided with articles dealing with the theme of the course to give the background knowledge that they needed to begin their experiments. Later they were allowed to choose articles from peer-reviewed journals based on their own scientific interests. Again, this allowed the students to feel senses of ownership over what they were learning and to feel invested in the course. There were weekly quizzes based on class discussions to assess students' comprehension of both the techniques being used and the topics being explored. These were generally take-home and require a significant amount of research to answer the questions. (There was not a text to study for these quizzes, but rather a problem-solving format.) Students also participated in weekly laboratory meetings where they presented their data orally to both the instructor and the student peers. They were graded on their preparation and ability to communicate to the audience. An additional oral assignment resembling a journal club was also given. Here a laboratory group prepared a scientific paper to present informally to the entire class. The final assessment involved a laboratory notebook, a written manuscript, and a formal oral presentation. The manuscript was submitted following the “Instructions to Authors” provided by the Journal of Biological Chemistry. Essentially, the students prepared a manuscript for publication based on their laboratory results. The oral presentation was structured as a formal seminar given to a scientific audience. Faculty and students from the science departments were invited and the talk was based upon background information and data generated during the semester.

Expenses—

Creativity was the key in providing resources and supplies for these two courses. The instructors routinely shared equipment and reagents including enzymes. A Davis Grant from the Colleges of the Fenway initially funded the “Advanced Biochemistry” course, allowing the purchase of equipment required to set up a biochemistry laboratory and to carry out protein purification. In addition, collaborating Colleges of the Fenway institutions contributed to special items, for example, the purchase of an immunological kit.

While these courses can be expensive, it should be noted that “Advanced Biochemistry” was initially established with a small grant ($5,000) that was primarily used to buy equipment for SDS-PAGE and basic laboratory supplies such as automatic pipettors. Sharing of equipment between the Biology and Chemistry department is routinely done, as for example with the sonicator and centrifuges, which helps to defray expenses. Some cost cutting has already been done in “Molecular Biology” by using restriction enzymes for cloning rather than the newer and considerably more expensive topoisomerase cloning vectors. Kits are convenient but not crucial. For example, plasmid DNA can be isolated using the boiling method or by alkaline lysis without using columns. Students in “Molecular Biology” used the boiling method followed by phenol/chloroform extraction and ethanol precipitation to isolate plasmid DNA, which they compared with DNA isolated using a Qiagen kit for yield and purity. A way to cut costs in “Advanced Biochemistry” is to use commercially available plasmids and purify a protein encoded by that plasmid. Currently, one of the proteins being used this year is GFP (green fluorescent protein), purchased at substantial educational discount from Bio-Rad (Hercules, CA) as part of a student kit. Whatever the resources, it is important for students to get hands-on laboratory experience and to be invested in their projects.

Commercial reagents, such as GST vectors, glutathione Sepharose 4B, Qiagen kits, etc., were purchased using funds from the Biology and Chemistry departments specifically set aside for the courses. However, in order to prepare students for what they will encounter in a typical research laboratory, the instructors felt it was important to use molecular biology kits. The majority of Simmons graduates will work in a research laboratory at some point in their careers, whether they are employed in one of the many research institutions or biotechnology companies in Boston and Cambridge or during graduate school. The Qiagen kits are used everywhere and are very user-friendly, justifying their cost. The cloning success of the “Molecular Biology” laboratory depended on having large quantities of pure DNA.

Also important were the shared resources of both the Chemistry and Biology departments. These courses took advantage of the overlap of techniques to purchase supplies and materials together. While the initial cost was significant, the subsequent costs can be modified to suit any budget. Additionally, techniques chosen to demonstrate advanced topics could be selected based on funds available at a given institution. It is less a matter of what experiments are done but more of how they are carried out. These courses need not be expensive but require some creativity on the part of the faculty. Using joint budgets from two departments helps to defray costs. Taking advantage of corporate willingness to help in the education of undergraduate scientists is another avenue that can be explored and utilized as described here.

CONCLUSION

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. Acknowledgements
  7. REFERENCES

The students clearly benefited from completing both the “Molecular Biology” and “Advanced Biochemistry” courses. With their emphasis on analyzing and doing experiments, these courses prepared the students for further research experience whether it is in graduate school or in a research laboratory. They also learned the material in greater depth than they would in a purely lecture-based approach because of the extensive use of journal articles. The format of these two courses also allowed the instructors opportunities to go beyond what is in the textbook and to demonstrate the real world application of the knowledge gained. “Advanced Biochemistry,” with its exclusive laboratory-based format, provided an excellent transition for continued research in both academic and biotechnology settings as attested by former students.

Table Table I. Comparison of course content
 “Molecular Biology”“Advanced Biochemistry”
Focus of laboratory workInstructor drivenStudent driven
Type of laboratory projectsCloning of a chromosomal gene into a protein expression vector to generate a tagged proteinPurification of a tagged protein; choice of tagged proteins offered
Laboratory techniquesIntroduction to key techniquesApplication of key techniques
Student assessmentCritical analysis of recent literatureUse of journal club to discuss recent literature
 Write a review of a recently published manuscriptProduce and write a manuscript
 Exams and quizzesOral seminar presentation of research results
 Laboratory reports 

Acknowledgements

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. Acknowledgements
  7. REFERENCES

We thank the Colleges of the Fenway, Davis Educational Foundation for the initial funding for the Advanced Biochemistry course. We are grateful to Qiagen, Inc. for the generous donation of the protein expression vector, pQE31, and to Amersham Pharmacia Biotech, Inc. for donating the laboratory manuals and supplies.

  • 1

    The abbreviations used are: GST, glutathione S-transferase; IPTG, isopropyl β-D-thiogalactoside.

REFERENCES

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. Acknowledgements
  7. REFERENCES
  • 1
    E. J. Wood (2001) Biochemistry and molecular biology teaching over the past 50 years, Nat. Rev. Mol. Cell. Biol. 2, 217221.
  • 2
    R. Boyer (2003) Concepts and skills in the biochemistry/molecular biology lab, Biochem. Mol. Biol. Educ. 31, 102105.
  • 3
    E. Bell (2001) The future of education in the molecular life sciences, Nat. Rev. Mol. Cell. Biol. 2, 221225.
  • 4
    Pharmacia Biotech (1997) GST Gene Fusion System 3rd Ed., Revision 2, Amersham Pharmacia Biotech Inc., Piscataway, NJ.
  • 5
    N. Erickson, J. C. Phipps, J. Lopilato, J. A. Roecklein-Canfield (2002) Purification of the Escherichia coli antisilencer protein BglJ by the GST fusion system, J. Undergrad. Chem. Res. 3, 131136.