Proteins as Products

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Chapter 4

• Proteins as Products

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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

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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

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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

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Central Dogma

• DNA codes for RNA which codes for proteins.

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Translation

• A protein is a string of amino acids held
  together by peptide bonds and do most of the work
  in a cell

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Translation
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Translation
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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 its intended
  function.
• The protein must fold into its appropriate
  3-dimensional shape.

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Protein Structure

• Proper folding of the protein is essential for
  its activity because it must bind its substrate
  to perform its job.

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Protein Structure

• Primary Peptide bonds in a chain of amino acids
• Secondary Hydrogen bonding between amino acids
  forms alpha-helices and beta-sheets
• Tertiary three dimensional folding of protein
  due to disulfide linkages and hydrophobic
  interactions between alpha-helices and
  beta-sheets
• Quaternary aggregation of multiple polypeptide
  chains

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Protein Structure

• Glycosylation
• Carbohydrate units added to protein
• Increases solubility, orients protein in
  membrane, extends life of protein
• Occurs in the golgi

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Protein Structure
5-GATCTGAATCGCTATGGC-3
3-CTAGACTTAGCGATACCG-5 mRNA
5-GAUCUGAAUCGCUAUGGC-3
CUAGACUUAGCGAUACCG Asp,
Leu, Asn, Arg, Tyr, Gly
Coding Template mRNA tRNA amino acid
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Protein Structure

• DNA codes for proteins that confer traits

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Protein Engineering

• Directed Molecular Evolution
• Introducing specific, predefined alterations in
  the DNA sequence.

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Protein Production

• Steps in bioprocessing

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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

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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

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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

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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

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Protein Extraction

• The target protein must be separated from the
  complex mixture of biological molecules

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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

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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
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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

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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

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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

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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

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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

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Protein Purification

• High Performance Liquid Chromatography (HPLC)
• Applies high pressure to drive sample through the
  column faster

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Protein Verification

• SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)
• Separates proteins in an electrical field based
  on molecular size

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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
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Protein Verification

• Polyacrylamide Gel Electrophoresis (PAGE)
• Much tighter gel matrix than agarose, which makes
  polyacrylamide ideal for separating proteins

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Protein Verification

• SDS-PAGE to test for purity

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Protein Verification
SDS-PAGE Animation
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Application

• Recombinant human insulin

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Preserving Proteins

• Lyophilization (freeze drying)
• Placed under vacuum to hasten evaporation of
  water
• Containers are sealed after water is removed

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Scale-up of Protein Purification

• RD 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

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Postpurification Analysis

• Protein Sequencing
• Determining the order of amino acids
• X-ray Crystallography
• Determining tertiary and quaternary structure of
  protein

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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