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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

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chapter 4

Chapter 4

Proteins as Products

proteins as biotech 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 products1
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 products2
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
Central Dogma
  • DNA codes for RNA which codes for proteins.
translation
Translation
  • A protein is a string of amino acids held together by peptide bonds and do most of the work in a cell
protein structure
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 structure1
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 structure2
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
protein structure3
Protein Structure
  • Glycosylation
    • Carbohydrate units added to protein
    • Increases solubility, orients protein in membrane, extends life of protein
    • Occurs in the golgi
protein structure4
Protein Structure

Coding:

Template:

mRNA:

tRNA:

amino acid:

5’-GATCTGAATCGCTATGGC-3’

3’-CTAGACTTAGCGATACCG-5’

mRNA 5’-GAUCUGAAUCGCUAUGGC-3’

CUAGACUUAGCGAUACCG

Asp, Leu, Asn, Arg, Tyr,Gly

protein structure5
Protein Structure
  • DNA codes for proteins that confer traits
protein engineering
Protein Engineering
  • Directed Molecular Evolution
    • Introducing specific, predefined alterations in the DNA sequence.
protein production
Protein Production
  • Steps in bioprocessing
protein expression
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 expression1
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 expression2
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 expression3
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
Protein Extraction
  • The target protein must be separated from the complex mixture of biological molecules
protein extraction1
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
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 purification1
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 purification2
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 purification3
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 purification4
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 purification5
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 purification6
Protein Purification
  • High Performance Liquid Chromatography (HPLC)
    • Applies high pressure to drive sample through the column faster
protein verification
Protein Verification
  • SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)
    • Separates proteins in an electrical field based on molecular size
protein verification1
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 verification2
Protein Verification
  • Polyacrylamide Gel Electrophoresis (PAGE)
    • Much tighter gel matrix than agarose, which makes polyacrylamide ideal for separating proteins
protein verification3
Protein Verification
  • SDS-PAGE to test for purity
protein verification4
Protein Verification

SDS-PAGE Animation

application
Application
  • Recombinant human insulin
preserving proteins
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
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
Postpurification Analysis
  • Protein Sequencing
    • Determining the order of amino acids
  • X-ray Crystallography
    • Determining tertiary and quaternary structure of protein
proteomics
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