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 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?) me.asphttp:// me.asp 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 100kD Smaller the protein higher the % gel used Laemmli 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 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