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BIO403: Biotechnology

Unit 4: Protein Engineering and Proteomics   A proteome is the collection of all proteins expressed by a genome, and proteomics analyzes the proteome.  Proteins serve almost every purpose imaginable, from catalyzing reactions to providing structural support.  Research into proteomics can be more complex than genomics, as they are inherently much more versatile biomolecules.  
           
This unit will summarize the current state of proteomics and protein engineering.  We will first look at how a protein is currently studied, such as isolating and characterizing a protein.  We will then look at engineering novel proteins based on current techniques and knowledge.

Unit 4 Time Advisory
This unit should take you approximately 18 hours to complete.

☐    Subunit 4.1: 1.5 hours

☐    Subunit 4.2: 2.5 hours

☐    Subunit 4.3: 2.0 hours

☐    Subunit 4.4: 2.5 hours

☐    Subunit 4.5: 3.5 hours

☐    Subunit 4.6: 6.0 hours

☐    Subunit 4.6.1: 0.5 hour

☐    Subunit 4.6.2: 1.0 hour

☐    Subunit 4.6.3: 1.0 hour

☐    Subunit 4.6.4: 0.5 hour

☐    Subunit 4.6.5: 0.5 hour

☐    Subunit 4.6.6: 0.5 hour

☐    Subunit 4.6.7: 1.0 hour

☐    Subunit 4.6.8: 1.0 hour

Unit4 Learning Outcomes
Upon successful completion of this unit, the student will be able to:

  • Describe how proteins are isolated, characterized, and sequenced.
  • Compare and contrast protein interaction techniques.
  • Discuss and contrast the design of recombinant proteins expression in prokaryotes and eukaryotes.
  • Identify challenges during the production of biologically active proteins.
  • Describe novel function-oriented protein design.

4.1 Protein Visualization and Identification   4.1.1 SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)   - Reading: Davidson College: Department of Biology’s “SDS-PAGE (PolyAcrylamide Gel Electrophoresis)” Links: Davidson College: Department of Biology’s  “SDS-PAGE (PolyAcrylamide Gel Electrophoresis)” (HTML)
 
Instructions: Please study this page.  Please note that electrophoresis can be used to separate substances, which have electrical charge.  In the SDS-PAGE technique, proteins are associated with SDS, thus they become negatively charged.  Furthermore, the smaller SDS-associated proteins will travel faster in the electric field than the larger ones.   
 
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4.1.2 Western Blot   - Reading: Davidson College: Department of Biology’s “Western Blot Procedure” Links: Davidson College: Department of Biology’s “Western Blot Procedure” (HTML)
 
Instructions: Please study the outline of the Western blot analysis.  This procedure is also called immunoblot technique, because it is utilizing antibodies to detect specific proteins in a mixture.  In reality, antibodies are not absolutely specific, thus this method may produce a false positive or negative signal.
 
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  • Lecture: Journal of Visualized Experiments: Gallagher and Chakavarti’ s “Immunoblot Analysis” Link: Journal of Visualized Experiments:  Gallagher and Chakavarti’ s “Immunoblot Analysis” (Adobe Flash)
     
    Instructions: Please watch the video (16 minutes) for a step-by-step tutorial on the immunoblot technique.
     
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  • Assessment: The University of Arizona: The Biology Project’s “Introduction to Western Blot Activity” Link: The University of Arizona:  The Biology Project’s “Introduction to Western Blot Activity” (HTML)
     
    Instructions:  In order to assess your knowledge, follow the instructions and complete the four problems at the end of this activity.  Answer key is provided.  You can progress page by page by clicking on the “Next” bottom toward the bottom of the page.
     
    Note: One animation requires a QuickTime plugin.
     
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4.2 Protein Isolation and Purification   4.2.1 HPLC (High-Performance Liquid Chromatography)   - Reading: Jim Clark’s ChemGuide: “High-Performance Liquid Chromatography—HPLC” Link: Jim Clark’s ChemGuide: “High-Performance Liquid Chromatography—HPLC” (HTML)
 
Instructions: Please study this page.  Please note that HPLC is used to purify peptides; it is not a typical protein purification method.
 
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4.2.2 Affinity Chromatography   - Reading: John W. Kimball's Biology Pages: “Affinity Chromatography” Link: John W. Kimball's Biology Pages:  “Affinity Chromatography” (HTML)
 
Instructions: Please study this page.  Please note that affinity chromatography is a very efficient purification technique as long as the immobilized molecule binds strongly and specifically to the protein of interest.  Antibody-antigen interaction is an example of strong and specific binding.
 
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  • Reading: Davidson College: Department of Biology’s “Affinity Chromatography Method” Link: Davidson College: Department of Biology’s “Affinity Chromatography Method” (HTML)
     
    Instructions: Please study the outline of the Affinity Chromatography Method.
     
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4.2.3 Ion Exchange Chromatography   - Reading: Florida State University: Michael Blaber’s “Protein Purification: Column Chromatography—Ion Exchange; Dialysis and Concentration” Link: Florida State University:  Michael Blaber’s “Protein Purification: Column Chromatography—Ion Exchange; Dialysis and Concentration” (HTML)
 
Instructions: Please study this page.  Please note that proteins are typically purified from a biological sample.  Ion exchange chromatography is a low efficiency, but inexpensive purification technique.  It is oftentimes employed to remove substances from a complex mixture, which would otherwise interfere with the following specific purification step.
 
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4.3 Protein Sequencing   4.3.1 Edman Degradation   - Reading: Wikibooks’ “Protein Primary Structure” Link: Wikibooks’ “Protein Primary Structure” (PDF)
 
Instructions: Please learn the “Edman Degradation” section on this page.
 
Terms of Use: The article above is released under a Creative Commons Attribution-Share-Alike License 3.0 (HTML).  You can find the original Wikibooks version of this article here (HTML).

  • Lecture: YouTube: Lamechivanes’ “Sequence Determination One Terminal Unit at a Time” Link: Video Lecture: YouTube: Lamechivanes’ “Sequence Determination One Terminal Unit at a Time” (YouTube)
     
    Instructions: Please watch the video (3 minutes) for a step-by-step tutorial on Edman degradation.
     
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4.3.2 Mass Spectrometry   - Reading: The University of Leeds: Alison Ashcroft’s “An Introduction to Mass Spectrometry” Link: The University of Leeds:  Alison Ashcroft’s “An Introduction to Mass Spectrometry” (HTML)
 
Instructions: Please study sections “1. What Is Mass Spectrometry (MS)? What Information Does Mass Spectrometry Provide?” and the content of subsections “4.1 Introduction,” “8.1 Tandem Mass Spectrometry,” “8.2 Tandem Mass Spectrometry Analyses,” and “8.3 Peptide Sequencing by Tandem Mass Spectrometry “ on this page.
 
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  • Assessment: The Saylor Foundation: "BIO403 Unit 4.3 Assessment" Link: The Saylor Foundation: "BIO403 Unit 4.3 Assessment" (HTML)
     
    Instructions: You will find link to the "Mass Spectrometry" assessment on this page. This is a multiple choice assessment with one correct answer. Clicking on an answer will bring you to another page. If your answer is correct, then it is acknowledged with a short explanation. Please read the explanation carefully. If you clicked on the wrong answer, then the click will bring you to a tutorial page. Please study the tutorial page carefully. You will be prompted to return to the assessment and complete it again. Please note that mass spectrometry is a widely accepted confirmatory technique for the identification of organic molecules.

4.4 Protein Interaction Methods   4.4.1 Yeast Two-Hybrid System   - Reading: John W. Kimball’s Biology Pages: “Proteomics” Link: John W. Kimball’s Biology Pages:  “Proteomics” (HTML)
 
Instructions: Please study “The Yeast Two-Hybrid System” section on this page.
 
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4.4.2 Co-Immunoprecipitation   - Reading: National Center for Biotechnology Information’s Bookshelf: Garland Science: Alberts, et al.’s Molecular Biology of the Cell, 4th edition: “Analyzing Protein Structure and Function” Link:  National Center for Biotechnology Information’s Bookshelf: Garland Science: Alberts, et al.’s Molecular Biology of the Cell, 4th edition:  Analyzing Protein Structure and Function” (HTML)
 
Instructions: Please study "Affinity Chromatography and Immunoprecipitation Allow Identification of Associated Proteins" section on this page.
 
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4.4.3 Protein Arrays   - Reading: Functional Genomics: Mike Taussig and Oda Stoevesandt’s “Protein Arrays Resource Page” Link: Functional Genomics:  Mike Taussig and Oda Stoevesandt’s “Protein Arrays Resource Page” (HTML)
 
Instructions: Please study this page.  Please note that there are different types of protein arrays.  Protein arrays represent a more recent development of high throughput screening compared to DNA microarrays. 
 
 
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4.5 Recombinant Proteins   4.5.1 Expression by Bacteria   - Reading: European Molecular Biology Laboratory’s “Protein Expression and Purification Core Facility: Protein Expression E. coli” Link: European Molecular Biology Laboratory’s "Protein Expression and Purification Core Facility: Protein Expression E. coli” (HTML)
 
Instructions: Please study this page.  Please note that E. coli can be employed to express a functional protein only if the biological function of the protein does not require posttranslational modification.
 
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4.5.2 Expression by Yeast   - Reading: John W. Kimball’s Biology Pages: “Gene Regulation in Eukaryotes” Link: John W. Kimball’s Biology Pages: “Gene Regulation in Eukaryotes” (HTML)
 
Instruction: Please study this page.  Focus on the differences between eukaryotic and prokaryotic genes expression, including posttranslational modifications.
 
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  • Reading: BioPharm International: Rudolph et al.’s “Expression of Recombinant Proteins in Yeast” Links: BioPharm International: Rudolph et al.’s “Expression of Recombinant Proteins in Yeast” (HTML)
     
    Instructions: Please study this page.  Please note that Pichia pastoris and other yeast hosts have the capability to modify recombinant proteins posttranslationally.  Posttranslational modification is an essential feature of biologically active proteins.
     
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4.5.3 Expression by Mammalian Cells   - Reading: BioMed Central: BMC Biotechnology: Nehlsen et al.’s “Recombinant Protein Expression by Targeting Pre-Selected Chromosomal Loci” Link: BioMed Central: BMC Biotechnology:  Nehlsen et al.’s “Recombinant Protein Expression by Targeting Pre-Selected Chromosomal Loci”  (HTML or PDF)
 
Instructions: Please read the “Background” section found under “Abstract” on this page.  You can access the PDF version from the righthand side of the page, under "viewing options."  This is a peer-reviewed publication.
 
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4.5.4 Protein Glycosylation   - Reading: National Center for Biotechnology Information's Bookshelf: Lodish, Berk, Zipursky, et al.’s Molecular Cell Biology, 4th edition: “Protein Glycosylation in the ER and in Golgi Complex” Link: National Center for Biotechnology Information's Bookshelf:  Lodish, Berk, Zipursky, et al.’s Molecular Cell Biology, 4th edition:  “Protein Glycosylation in the ER and in Golgi Complex” (HTML)
 
Instructions: Please study this entire section from Lodish, Berk, Zipurksy, et al.’s Molecular Cell Biology textbook.  Please note that protein glycosylation is very important for certain proteins to function properly in eukaryotes.  Proteins that are expressed in prokaryotic cell, for example, in E. coli, cannot be glycosylated, because these cells lack membrane-bound subcellular organelles.

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4.6 Protein Engineering   4.6.1 Protein Folding in Biotechnology   - Reading: Scientific Electronic Library Online Brazil: Brazilian Journal of Medical and Biological Research: J.M. Yon’s “Protein Folding: A Perspective for Biology, Medicine and Biotechnology” Link: Scientific Electronic Library Online Brazil: Brazilian Journal of Medical and Biological Research: J.M. Yon’s “Protein Folding: A Perspective for Biology, Medicine and Biotechnology” (HTML or PDF)
 
Instructions: Please study the content of “Protein Folding in Biotechnology: Protein Engineering and Design” on this page.  You can access the PDF version from the right hand side of the page.  Author Yon works at the Institut de Biochimie, Biophysique Moléculaire et Cellulaire, UMR CNRS, Université de Paris-Sud, Orsay, France.
 
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4.6.2 Enzyme Binding Site Specificity   - Reading: Oxford University Press’s Journal of Experimental Biology: Jeanneau, et al.’s “Manipulating PEPC Levels in Plants” Links: Oxford University Press’s Journal of Experimental Biology: Jeanneau, et al.’s “Manipulating PEPC Levels in Plants” (HTML or PDF)
 
Instructions: Please study this article.  Review what you have learned about RiBisCo, and focus on the “Transgenic Plants” section on this page.  You can access the PDF version by clicking the “Full Text (PDF)” button on the right-hand side of the page.  This is a peer-reviewed publication.
 
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4.6.3 Structural Scaffolds and Domain Recombination   - Reading: Oxford University Press’s Nucleic Acids Research: Sleight et al.’s “In-Fusion BioBrick Assembly and Re-Engineering” Links: Oxford University Press’s Nucleic Acids Research:  Sleight et al.’s “In-Fusion BioBrick Assembly and Re-Engineering” (HTML or PDF)
 
Instructions: Please study this entire article. Focus on the “Introduction,” “Results,” and “Discussion” sections.  You can access the PDF from the right-hand side of the page.  This is a peer-reviewed publication. The authors work at the Department of Bioengineering, University of Washington, Seattle.
 
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4.6.4 Nonnatural Amino Acids   - Reading: The FASEB Journal: Budisa, et al.’s “Toward the Experimental Codon Reassignment In Vivo: Protein Building With an Expanded Amino Acid Repertoire” Link: The FASEB Journal:  Budisa, et al.’s “Toward the Experimental Codon Reassignment In Vivo: Protein Building With an Expanded Amino Acid Repertoire” (HTML or PDF)
 
Instructions: Please focus on the " 'Restricted' vs. 'Relaxed' Genetic Code" and "Codon Reassignment In Living Cell" sections.  You can access the PDF from the right-hand side of the page.  The authors work at the Max Planck Institut für Biochemie, Germany.  This is a peer-reviewed publication.
 
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4.6.5 Domain Recombination   - Reading: BioMed Central: BMC Medicine: David Davis and David Stokoe’s “Zinc Finger Nucleases as Tools To Understand and Treat Human Diseases” Link: BioMed Central:  BMC Medicine:  David Davis and David Stokoe’s “Zinc Finger Nucleases as Tools To Understand and Treat Human Diseases” (HTML or PDF)
 
Instructions: Please focus on the following sections: “Methods for Design, Testing and Implementation of Zinc Finger Proteins (ZFPs):” “Addition of Functional Domains Expands the Utility of ZFPs:” and “Addition of Eendonuclease Activities to ZFPs to Create Targeted DNA Scissors.”  Please note that domain recombination is the recombining of two known functional domains of proteins to create a new kind of protein.  You can access the PDF from the right-hand side of the page.  Authors work in the Department of Molecular Biology at Genentech Inc.  This is a peer-reviewed publication.
 
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4.6.6 DNA Shuffling   - Reading: National Center for Biotechnology Information’s Bookshelf: Wiley-Liss: Strachan and Read’s Human Molecular Genetics, 2nd edition: “Chapter 14: Our Place in the Tree of Life” Link: National Center for Biotechnology Information’s Bookshelf:  Wiley-Liss: Strachan and Read’s Human Molecular Genetics, 2nd edition:  “Chapter 14: Our Place in the Tree of Life” (HTML)
 
Instructions: Please study the “14.5.2 Exon Shuffling Permits Diverse Combinations of Structure and Functional Modules, and May be Mediated by Transposable Elements” section on this page.
 
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4.6.7 Combinatory Protein Libraries   - Reading: National Center for Biotechnology Information’s PubMed: PLoS Biology: David R. Halpin and Pehr B. Harbury’s “DNA Display II. Genetic Manipulation of Combinatorial Chemistry Libraries for Small-Molecule Evolution” Link: National Center for Biotechnology Information’s PubMed:  PLoS Biology:  David R. Halpin and Pehr B. Harbury’s “DNA Display II. Genetic Manipulation of Combinatorial Chemistry Libraries for Small-Molecule Evolution” (HTML or PDF)
 
Instructions: Please study “Strategy” in the “Results” section.  You can access the PDF from the top right corner of the page.  The two authors, Halpin and Harbury, work in the Department of Biochemistry at Stanford University’s School of Medicine. This is a peer-reviewed publication.
 
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4.6.8 Biomaterials   - Reading: National Center for Biotechnology Information’s Bookshelf: Harvard Stem Cell Institute: Stephanie M. Willerth and Shelly E. Sakiyama-Elbert’s StemBook: “Combining Stem Cells and Biomaterial Scaffolds for Constructing Tissues and Cell Delivery” Link:  National Center for Biotechnology Information’s Bookshelf: Harvard Stem Cell Institute: Stephanie M. Willerth and Shelly E. Sakiyama-Elbert’s StemBook:Combining Stem Cells and Biomaterial Scaffolds for Constructing Tissues and Cell Delivery” (HTML or PDF)
 
Instructions:  Please study this page.  You can access the PDF from the right-hand side of the page under the “Download” section. The two authors, Willerth and Sakiyama-Elbert, are affiliated with the Department of Biomedical Engineering at Washington University, St. Louis.
 
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