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Chapter 4
Proteins as Products
Proteins as Biotech Products
Enzymes – speed up chemical reactions
• Synthesis – combines small molecules to make larger
molecules
• ATP synthetase, peptidyl transferase, polymerase
• Depolymerization – breaks down large molecules
• Amylase, lipase, protease
Hormones – carry chemical messages
Antibodies – part of immune response
Proteins as Biotech Products
Therapeutic Protein
• used to treat a disease that is caused by a gene that
fails to produce a necessary protein or that produces
a dysfunctional protein
Proteins as Biotech Products
Food processing – the creamy in ice cream
Textile and leather goods – bio-bleaches
Detergents – enzymes to dissolve stains
Paper manufacturing and recycling – reduce
negative environmental impacts
Adhesives – barnacles and mussels
Bioremediation – proteins used to clean up
harmful waste
Central Dogma
DNA codes for RNA which codes for proteins.
Translation
A protein is a string of amino acids held
together by peptide bonds and do most of
the work in a cell
Translation
Translation
Protein Structure
 Once the amino acid chain is
released from the ribosome, a
number of modifications are
made in order for the protein
to perform it’s intended
function.
 The protein must fold into it’s
appropriate 3-dimensional
shape.
Protein Structure
Proper folding of the protein is essential for it’s
activity because it must bind it’s substrate to
perform it’s job.
Protein Structure
 Primary – Peptide bonds in a chain of
amino acids
 Secondary – Hydrogen bonding
between amino acids forms alphahelices and beta-sheets
 Tertiary – three dimensional folding
of protein due to disulfide linkages
and hydrophobic interactions
between alpha-helices and betasheets
 Quaternary – aggregation of multiple
polypeptide chains
Protein Structure
Glycosylation
• Carbohydrate units added to protein
• Increases solubility, orients protein in membrane,
extends life of protein
• Occurs in the golgi
Protein Structure
5’-GATCTGAATCGCTATGGC-3’
Coding:
Template: 3’-CTAGACTTAGCGATACCG-5’
mRNA:
mRNA 5’-GAUCUGAAUCGCUAUGGC-3’
tRNA:
CUAGACUUAGCGAUACCG
amino acid:
Asp,
Leu,
Asn,
Arg,
Tyr,
Gly
Protein Structure
DNA codes for proteins that confer traits
Protein Engineering
Directed Molecular Evolution
• Introducing specific, predefined alterations in the
DNA sequence.
Protein Production
Steps in bioprocessing
Protein Expression
Bacteria
 Advantages
 Cheap and easy to grow
 Biology is well-defined
 High yield of recombinant proteins in culture
 Disadvantages
• Many proteins become insoluble in inclusion bodies
• Most if not all post-translational modifications are not added
Protein Expression
Fungi
 Advantages
• Grown in simple, inexpensive media
• Secrete many proteins into the media
• Capable of many post-translational modifications
 Disadvantages
• Recombinant proteins usually expressed at low levels
• Some post-translational modifications differ significantly
Protein Expression
Plants
 Advantages
• Rapid growth and reproductive rates
• Perform most post-translational modifications
• Transgenic plants can be self-fertilized
 Disadvantages
• Not all mammalian proteins are expressed in plants
• Plant cells have a tough cell wall
• Some plants produce proteins in their green leaf tissues
Protein Expression
Mammalian Cell Culture
 Advantages
• Protein-folding and post-translational modification
• Powerful promoters to regulate protein expression
• High expression levels
 Disadvantages
• Complex and expensive nutritional requirements
• Slow growing
Protein Extraction
The target protein must be separated from the
complex mixture of biological molecules
Protein Extraction
Isolated proteins must be stabilized
 Very sensitive to changes in temperature
 Proteases that could digest the target protein are a
threat
 Protein folding is dependent on the pH of the
environment
Protein Purification
Chromatography
 A method to separate proteins by size, charge, or
chemical properties as they pass through a column of
resin beads
Chromatography
Animation
Protein Purification
 Chromatography
 Resin/matrix – solid particles in the column
 Sample – protein mixture that is loaded on the column
 Elution – liquid that passes through the column and is collected
in fractions
Protein Purification
Size Exclusion Chromatography
 Separates proteins based on size
 Small molecules get caught in the beads
 Larger molecules pass quickly around the beads and elute first
Protein Purification
Ion Exchange Chromatography
 Separates molecules based on ionic charge
 Proteins are eluted by increasing the concentration of a salt
buffer
 Proteins with the weakest charge are eluted first
Protein Purification
Hydrophobic Interaction Chromatography
 Separates proteins based on repulsion to water
 Proteins are eluted by decreasing the salt concentration of the
buffer
 The least hydrophobic proteins are eluted first
Protein Purification
Affinity Chromatography
 Separates proteins based on molecular conformation
 Matrix is made of a ligand specific for the desired protein
 The protein is cleaved from the matrix using a site-specific
protease
Protein Purification
High Performance Liquid Chromatography (HPLC)
 Applies high pressure to drive sample through the
column faster
Protein Verification
SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)
 Separates proteins in an electrical field based on
molecular size
Protein Verification
 Sodium Dodecyl Sulfate (SDS)
 A detergent that denatures the secondary and tertiary structure of
the protein
 Coats the protein with negative charges
Add SDS
Protein Verification
Polyacrylamide Gel Electrophoresis (PAGE)
 Much tighter gel matrix than agarose, which makes
polyacrylamide ideal for separating proteins
Protein Verification
SDS-PAGE to test for purity
Protein Verification
SDS-PAGE Animation
Application
Recombinant human insulin
Preserving Proteins
Lyophilization (freeze drying)
• Placed under vacuum to hasten evaporation
of water
• Containers are sealed after water is removed
Scale-up of Protein Purification
R&D works on small scale
Large production demands protocols to scale-up
bioreactors
• If FDA approval has been gained for small-scale, cannot
change the parameters when scaled up
Postpurification Analysis
Protein Sequencing
• Determining the order of amino acids
X-ray Crystallography
• Determining tertiary and quaternary structure
of protein
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 Microarrays
• Identifies protein interactions