Download Introduction to Proteins: Biotech 2

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_ho
me.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



Stabilizing the Proteins in Solution


If intracellular, lyse the cells
Detergents or organic solvent can be used for lipid
membrane bound proteins
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



Centrifugation (sized based)
Filtration



Membrane, microfiltration, ultrafiltration
Diafiltration and dialysis
Chromatography


Ammonium sulfate
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
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


Protein chips


The variations of protein expression are then correlated to
onset or progression of a specific disease
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