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Introduction to Proteins as Products. Biotechnology 2. 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! - PowerPoint PPT Presentation
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Introduction to Proteins as Products
Biotechnology 2
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
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
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)
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
Basic components of a fermenter
Basic steps in bioprocessing
Lyophilization
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)
Process for making cheese
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)
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
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
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
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
Centrifugation
Filtration
Chromatography
Yield: % recovered from final product
Hydrophobic chromatography
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
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
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
Types of Chromatography Gel Filtration Chromatography: also called
size exclusion, molecules are separated according to their size and shape
Verification SDS-PAGE
Compare protein size to set of sizing standards run
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
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
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.
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)
Post-Purification Analysis Methods Protein Sequencing X-ray Crystallography
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
Protein Engineering Directed molecular evolution technology
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