Direct electrochemistry of proteins and enzymes: an introduction
Introduction to Proteins as Products
description
Transcript of Introduction to Proteins as Products
![Page 1: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/1.jpg)
Introduction to Proteins as Products
Biotechnology 2
![Page 2: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/2.jpg)
Review of the Basics Made up of amino acids Functions:
Regulatory role Structural support Transport There are literally thousands of functions and we
do not yet understand all of them! In order to understand their functions we have
to understand their structure
![Page 3: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/3.jpg)
Protein Structure Polymers of small units (amino acids) Proteins do NOT have a uniform structure
Due to 20 different amino acids available The chemical and physical properties are different
among the different amino acids Protein sequence reported by Frederick
Sanger in 1953. Protein folding determines structure
![Page 4: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/4.jpg)
Turning Proteins into Products Identifying proteins and their function is only
half the battle. Once identified, proteins typically need to be
grown and then purified and processed into usable, salable products.
Levels of Product Purity (least to most pure) Research grade Diagnostic grade Pharmaceutical grade (low to high dose)
![Page 5: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/5.jpg)
Examples of Purified Proteins Enzymes
Amylases, proteases, lipases (google the company, Genzyme-how many of the enzymes does this company make?)http://www.genzyme.com/business/biz_home.asp
http://www.genzymediagnostics.com/ Hormones Antibodies What was the first recombinant protein to
be mass produced? Cerezyme
![Page 6: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/6.jpg)
Basic components of a fermenter
![Page 7: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/7.jpg)
Basic steps in bioprocessing
Lyophilization
![Page 8: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/8.jpg)
More Examples of products: Food Processing (the creamy in ice cream) Textiles and Leather Goods (bio-stoning) Detergents (enzymes) Paper Manufacturing and Recycling Adhesives: Natural Glues Bioremediation: Treating Pollution with
Proteins (metallothioneins)
![Page 9: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/9.jpg)
Process for making cheese
![Page 10: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/10.jpg)
Protein Structures Levels of Organization
Primary (the AA sequence of its polypeptide chain)
Secondary (H bonding between peptide bonds) Tertiary (covalent, ionic, H bonding, hydrophobic) Quaternary (involves more than one subunit)
![Page 11: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/11.jpg)
![Page 12: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/12.jpg)
Protein Production
Upstream Processing: the actual expression of the protein in the cell Microorganisms- cheap, well understood, grow rapidly,
produce large amounts, clone in as cDNA, fusion gene (fusion protein), inclusion bodies, no glycosylation
Fungi – can do some posttranslational modifications Plants- 85% of current drugs from plants; rapid growth,
cheap, proteins not expressed properly Mammalian Cell Systems – finicky, grow slowly, and
expensive, BUT processes human proteins correctly Whole-animal –transgenic (goats making spider silk) Insect systems – baculoviruses are used as vector to
insert genes into insect cells
![Page 13: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/13.jpg)
Downstream Processing: the protein is separated from other parts of the cell and then isolated from other proteins
Preparing the Protein Extract for Purification If intracellular, lyse the cells Detergents or organic solvent can be used for lipid
membrane bound proteins Stabilizing the Proteins in Solution
Temperature, decrease protease activity and denaturing activity, maintain biological activity
Separating the Components in the Extract Utilize the chemical and physical properties of
proteins to separate them
![Page 14: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/14.jpg)
Stabilizing the Protein pH: extremes will denature the protein Temperature: thermal stability varies among
proteins Typically high temp more damaging A lot of protein purification happens at 0C or refrigerated
conditions Proteases and nucleases: degradative enzymes Adsorption surfaces: many proteins denatured by
contact w/air, water, glass, or plastic
![Page 15: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/15.jpg)
Protein precipitation Ammonium sulfate
Centrifugation (sized based) Filtration
Membrane, microfiltration, ultrafiltration Diafiltration and dialysis
Chromatography Size-exclusion, ion-exchange, affinity,
hydrophobic, iso-electric focusing, 2D electrophoresis
Analytic Methods HPLC, mass spectrometry
![Page 16: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/16.jpg)
Centrifugation
![Page 17: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/17.jpg)
Filtration
![Page 18: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/18.jpg)
Chromatography
Yield: % recovered from final product
![Page 19: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/19.jpg)
Hydrophobic chromatography
![Page 20: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/20.jpg)
Types of Chromatography
Ion Exchange: Charged molecules bind to oppositely charged group that been immobilized on the matrix
Hydrophobic Interaction Chromatography: non polar groups on the surface of proteins “interact” with the hydrophobic groups. Hydrophobic materials stick tightly together under high salt conditions
![Page 21: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/21.jpg)
Hydrophobic interaction chromatography In the Bio-Rad HIC kit:
Equilibration buffer prepares the column (2M ammonium sulfate buffer)
Elution buffers are low salt concentration (10mM Tris buffer)
Binding buffer: 4 M Ammonium sulfate buffer Buffers from high salt to low salt concentration
Binding, Equilibration, Wash, Elution buffer
![Page 22: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/22.jpg)
Types of Chromatography Affinity Chromatography: when an impure
protein solution is passed through this chromatographic material, the desired proteins binds to the immobilized ligand, where the other substances are washed through the column by a buffer
The material you want to capture “sticks” to the column and the rest is washed away
![Page 23: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/23.jpg)
Types of Chromatography Gel Filtration Chromatography: also called
size exclusion, molecules are separated according to their size and shape
![Page 24: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/24.jpg)
Verification SDS-PAGE
Compare protein size to set of sizing standards run
![Page 25: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/25.jpg)
SDS Page Electrophoresis process used for proteins: can
determine molecular weight of a protein The SDS (sodium dodecyl sulfate) helps to unfold
protein Materials Needed for SDS-PAGE
Molecular Weight Markers 4-20% acrylamide gradient gels Tris-glycine-SDS buffer Practice gel loading solution Marker protein Sample proteins Sealant 50 ml Coomassie Blue Staining Solution (silver stain is most sensitive) De-staining solution (7.5% acetic acid) and methanol
![Page 26: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/26.jpg)
SDS Page% Acrylamide gels based
on MW of protein 7% 50kD to 500kD 10% 20kD to 300kD 12% 10kD to 200kD 15-16% 3kD to 100kDSmaller the protein higher
the % gel usedLaemmli gels composed
of stacking and running gels at different pH
Process Unfolds the protein to
make it linear Separates the protein
and subunits by molecular weight
Coats the protein with negative charge (run like gel electrophoresis)
use of silver stain for SDS page - Google Videos
![Page 27: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/27.jpg)
![Page 28: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/28.jpg)
Preserving Proteins Lyophilization (freeze drying)
First frozen, placed under vacuum to hasten the evaporation of water (I.e. ice crystals go to water vapor). The containers are sealed after the water is removed, leaving the dried proteins behind.
![Page 29: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/29.jpg)
Scale-up of Protein Purification R&D starts with a small-scale level Production may demand a larger level
Small scale may not be adaptable If FDA approval has been gained for small-scale,
cannot change the parameters when scaled up (so scientists MUST make sure they can scale up before seeking approval)
![Page 30: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/30.jpg)
Post-Purification Analysis Methods Protein Sequencing X-ray Crystallography
![Page 31: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/31.jpg)
Proteomics Proteomes are compared under healthy and
diseased states The variations of protein expression are then correlated to
onset or progression of a specific disease Protein chips
Biochips that can be used to identify proteins Ways to test proteins
Chemical genetics (compare two same species organisms looking for presence and absence of protein)
Gene expression analysis: on/off switch Protein interaction analysis
![Page 32: Introduction to Proteins as Products](https://reader035.fdocuments.us/reader035/viewer/2022062521/56816065550346895dcf8f67/html5/thumbnails/32.jpg)
Protein Engineering Directed molecular evolution technology