Author Profile

Exploring eukaryotic protein structure and post-translational modifications.

Erica Jacobs, St. John's University

Location:

Abstract

This CURE provides opportunity for students to think and act as researchers by using computational, biochemical, and bioanalytical techniques to examine tick antigen proteins. Ticks are notorious vectors of viral, protozoan, and bacterial diseases, including Lyme disease. While an anti-vector vaccine capable of protecting people from diseases transmitted by a particular tick species is an alluring goal, only one such anti-tick vaccine is currently available. This vaccine targets Bm86, a protein from the midgut of Rhipicephalus microplus, a cattle tick. Not only does the vaccine limit parasitism of the cattle by ticks, data suggest that it can prevent transmission of tick-borne diseases including bovine anaplasmosis and babesiosis. However, similar vaccination approaches have not succeeded against ticks that transmit diseases to humans, and little is known about the antibody response to the antigen, or about the protein itself. Since the protein's structure and function are unknown, the research goal of this CURE is to purify Bm86 using an insect cell/baculovirus expression system and characterize it, including domain structure and post-translational modifications (glycosylation sites). There are homologs to Bm86 in every sequenced tick species examined, and future iterations of this CURE will characterize some of the homologs including those in Ixodes scapularis, the tick that is mainly responsible for transmitting Lyme in the eastern US, and Haemaphysalis longicornis, the Asian longhorned tick, a newly-discovered invasive species in the area that also has significant disease-transmitting potential. By understanding the structure and post-translational modifications of this protein, we hope to gain a better understanding of how to make effective anti-tick vaccines, including those for humans, that may prevent transmission of Lyme disease. Importantly, the basic parameters of this CURE can be used to examine other proteins besides tick antigens. For example, during the pandemic, the CURE pivoted from the tick antigen to the SARS-CoV-2 nucleocapsid protein, which was also expressed in an insect cell system. Instead of characterizing glycosylation sites, we characterized phosphorylation sites. It may be possible to use this same framework for many different eukaryotic proteins of research interest.

Student Goals

  1. Design methodology, procedures, practical workflows, and timelines for achieving experimental goals.
  2. Optimize experimental conditions, making and defending rational decisions during experimentation for how to proceed when trouble-shooting or interpreting ambiguous data. Revise initial workflows, timelines, and experimental conditions to adjust for preliminary results.
  3. Communicate about research progress orally, in writing, and in scientific poster format, and be able to articulate how project fits into a broader experimental and theoretical framework.

Research Goals

  1. Express and purify eukaryotic protein of interest (e.g., tick vaccine antigen) using an insect cell/baculovirus system.
  2. Characterize structural parameters of protein of interest, including domain layout and localization of post-translational modifications using enzymatic assays.

Context

This CURE is designed for upper-level chemistry, biology, and pharmacology students at a primarily undergraduate institution. We aim to involve students in research who might not otherwise have the opportunity by embedding a research opportunity into a regular biochemistry lab. The CURE focuses on tick proteins, but the approach could be adapted to many different proteins of interest. The CURE could also be adapted for a cell/molecular biology by focusing on cloning and protein expression, or for (bio) analytical chemistry instrumentation analysis lab by focusing on spectroscopic and mass spectrometric analysis.

Target Audience:Major
CURE Duration:A full term

CURE Design

The CURE is designed as a lab for upper-level students who are taking or have taken a one-semester introductory biochemistry course, but two semesters would be even better. It could also be adapted for cell/molecular biology or (bio) analytical chemistry instrumentation analysis labs. It has been taught for classes ranging from 12-24 students. The labs are divided into modules each occupying one or two three-hour lab sessions. Modules are typical of the types of methodology and reasoning that researchers encounter in biochemistry. Topics include macromolecular structure of proteins and carbohydrates and enzymatic catalysis. Skills developed include computational analysis of structure/post-translational modifications, and analytical techniques for protein characterization. Techniques include protein purification, analysis of concentration and purity, SDS-PAGE gels, including staining and quantitation, enzymatic assays, and mass spectrometric analysis. Computational tools utilized include BLAST, NCBI Conserved Domain Database (CDD)/SMART, PROSITE, PeptideCutter, Expasy Peptide Mass/pI, ClustalW, TMPred/SignalP, NetNglyc/NetOglyc, and MODELLER. Labs are structured with prelab assignments coordinated with the lectures to introduce students to concepts and their in-lab applications. New techniques are demonstrated in class.


The CURE differs from a traditional biochemistry lab course in that it has a unified theme of expressing and characterizing Bm86 homologs. While Bm86 has been used as an antigen in a successful anti-tick vaccine, the structure, function, and localization of its post-translational modifications are unknown. Students contribute to understanding and perhaps helping to solve a problem of biomedical importance, namely transmission of tick-borne disease. The CURE begins with an expression plasmid for Bm86 homologs in a bacterial vector. Before the course, the plasmid is used to generate baculovirus and infect host insect cells, causing them to express the protein and secrete it into the media. The his-tagged protein can then be purified from the media by standard immobilized metal affinity chromatography. Insect cells and baculovirus are not harmful to humans, and can be worked with under BSL1 conditions unless the baculovirus has been specifically engineered to infect mammalian cells. Although this eukaryotic expression system is more cumbersome and often lower yield than bacterial protein expression, it enables the characterization of eukaryotic proteins that cannot be correctly expressed, folded, and/or modified in bacteria, including secreted proteins, disulfide bonded proteins, and proteins bearing certain post-translational modifications.

Core Competencies: Analyzing and interpreting data, Planning and carrying out investigations
Nature of Research:

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Express and purify eukaryotic protein of interest (tick vaccine antigen) using an insect cell/baculovirus system.
Research Goal 2: Characterize structural parameters of protein of interest, including domain layout and localization of post-translational modifications using enzymatic assays.

Student Goal 1: Design methodology, procedures, practical workflows and timelines for achieving experimental goals.

Purify his-tagged protein of interest using immobilized metal affinity chromatography.

Predict structural parameters of protein of interest including pI/MW, domain organization, localization of glycosylation sites, etc using bioinformatics tools.


Student Goal 2: Optimize experimental conditions, making and defending rational decisions during experimentation for how to proceed when trouble-shooting or interpreting ambiguous data. Revise initial workflows, timelines, and experimental conditions to adjust for preliminary results.

Determine purity and absolute and relative yield of protein prep using SDS-PAGE analysis by reference to a protein standard via quantitation by the software package ImageJ.

Design and execute experiments to explore domain organization of protein via limited proteolysis, and compare results to computational predictions.


Student Goal 3: Communicate about research progress orally, in writing, and in scientific poster format, and be able to articulate how project fits into a broader experimental and theoretical framework.

Determine the concentration of protein prep using the colorimetric BCA assay, and compare results with gel analysis.

Design and execute experiments to localize glycosylation sites on the protein of interest using enzymatic deglycosylation, and compare with computational predictions.


Instructional Materials

Syllabus Syllabus for Biochemistry CURE lab.pdf (Acrobat (PDF) 1.9MB Aug27 21)

Protocols and references:

A. Computational Analysis
1. Predict parameters (MW, pI) from protein sequence (Expasy)
2. Predict domains (Blast CDD, SMART)
3. Predict post-translational modification sites (NetNglyc/NetOglyc)
4. Model structure (MODELLER)
5. Create figure with combined features (Excel, Powerpoint, Illustrator...)

Computational_Analysis.pdf (Acrobat (PDF) 114kB Aug23 21)

B. Purification
1. Invitrogen manual MAN0011581_HisPur_Cobalt_Resin_UG(1).pdf (Acrobat (PDF) 60kB Aug23 21)
2. Small scale purification protocol Small Scale his-tagged Protein Purification Protocol (Microsoft Word 2007 (.docx) 23kB Aug23 21) MAN0011581_HisPur_Cobalt_Resin_UG(1).pdf (Acrobat (PDF) 60kB Aug23 21)
3. Buffer exchange PAL_08.2414_CentrifugalDevice_SS.pdf (Acrobat (PDF) 774kB Aug23 21)
4. BCA assay manual MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf (Acrobat (PDF) 140kB Aug23 21)

C. Characterization
1. Protease domain analysis protocol https://pubmed.ncbi.nlm.nih.gov/16615907/
2. deglycosylation/in gel digestion protocol In gel Deglycosylation and Trypsin Digestion .docx (Microsoft Word 2007 (.docx) 66kB Aug23 21)
3. Crosslinking protocol DSS (disuccinimidyl suberate), No-Weigh™ Format.pdf (Acrobat (PDF) 410kB Aug23 21) DSS Crosslinking.docx (Microsoft Word 2007 (.docx) 23kB Aug23 21)
4. STAGE tip protocol (https://doi.org/10.1038/nprot.2007.261)

5. pI determination IEFgel_card_Novex.pdf (Acrobat (PDF) 63kB Aug23 21)Isoelectric focusing principles and procedure.pdf (Acrobat (PDF) 392kB Aug23 21)

Assessment

Example Rubric for Lab Reports (Acrobat (PDF) 54kB Aug23 21)
Example Rubric for Lab Notebooks (Acrobat (PDF) 69kB Aug23 21)
Rubric for Poster (Excel 2007 (.xlsx) 12kB Aug23 21)

Instructional Staffing

In addition to the primary instructor, the course has a TA. The TA has weekly pre-lab meetings with the primary instructor to familiarize them with the week's course materials, research goals, and learning objectives. The TA is also responsible for grading the lab notebooks.

Author Experience

Erica Jacobs, St. John's University

I would like to explore the CURE format for its potential to improve student outcomes and allow me to continue research during teaching-heavy semesters.


Advice for Implementation

1. Choose a cool protein—student buy-in is important.

2. Expect to focus on either purification or characterization. Doing both in a one-semester lab may not be realistic.

3. Insect cells and baculovirus are generally considered BSL1. They can be grown at room temperature without CO2 and do not require serum.

4. Most procedures do not require expensive reagents or equipment. For those that may, alternatives can be developed. For example:

  • If a UV/vis microplate reader is not available for the BCA assay, then students can get very decent measurements for the standards and samples by taking pictures of the tubes (200 ul PCR tubes work well) with their phones and analyzing intensity with the ImageJ free software package (https://imagej.nih.gov/ij/), which they will likely use anyways to quantitate protein bands on their Coomassie gels.
  • The protein digestion protocols to determine localization of post-translational modifications, etc., require mass spectrometric readout. In the absence of these specialized and expensive analytical instruments, consider reaching out to larger labs or resource centers. For some of these centers, outreach and education efforts are increasingly significant factors in their funding, and they may therefore be more receptive to collaborating than you might fear. Even if they can't give you a break on price, the cost for analyzing a protein in a gel band has gone down to ~$200 per band, and may fit in your budget. Make sure to talk to them before you prepare your samples.
  • Free platforms for academics for analysis of mass spectrometry data include MaxQuant (https://www.maxquant.org/) and the pLink/pFind suite (http://pfind.ict.ac.cn/software/pLink/).

Iteration

Goal 1: Protein purifications are notorious for requiring optimization. Parameters available for students to optimize include time, temperature, amount of affinity resin, and buffer/media conditions.
Goal 2: Again, enzymatic assays such as limited proteolysis and deglycosylation often require sometimes extensive optimization to be successful, including such parameters as choice of enzyme, amount of enzyme, buffer, incubation time/temperature, etc. The first time I taught this CURE, the deglycosylation results were inconsistent and confusing. Based on these results, the students repeated the experiment with adjusted parameters a few weeks later with much greater success. The students similarly ended up repeating the lectin affinity experiment, which required the elimination of the planned ELISA module.

Using CURE Data

Data produced by individual lab groups in the form of protocols for and results of the purification and characterization of the particular protein assigned to their group will be archived. Protocols and images will be preserved including on the online lab notebook platform Benchling. Lab partners will produce posters of their results which may be displayed at intramural or extramural poster sessions. Students will be acknowledged in any publications resulting from their work.

Resources

BactoBac Expression System Invitrogen_bactobacexpression.pdf (Acrobat (PDF) 556kB Aug27 21)
Producing Green Fluorescent Protein in Insect Cell Culture Using Baculovirus, A New
Laboratory Experience for UC Davis Biochemical Engineering Students

https://eelc.engineering.ucdavis.edu/producing-green-fluorescent-protein-in-insect-cell-culture-using-baculovirus-a-new-laboratory-experience-for-uc-davis-biochemical-engineering-students/

Dokudovskaya S, Williams R, Devos D, Sali A, Chait BT, Rout MP. Protease accessibility laddering: a proteomic tool for probing protein structure. Structure. 2006 Apr;14(4):653-60. doi: 10.1016/j.str.2006.02.006. PMID: 16615907.

STAGE tip peptide purification/desalting protocol: Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2, 1896–1906 (2007). https://doi.org/10.1038/nprot.2007.261