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