DNA is the Flash but Proteins are the Cash of Biotech

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DNA is the Flash but Proteins are the Cashof
Biotech
Ellyn Daugherty www.BiotechEd.com www.emcp.com/bio
tech www.sargentwelch.com/biotech AEEDaugher_at_aol.c
om 650-400-9424
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What are Proteins? Why are they so Important?

• Structure
• Large macromolecules (AA necklaces)
• Diff s and combos of 20 diff AA
• Functions
• Hormones, antibodies, pigments,
• transport, contractile, structural,
• recognition, and enzymes
• Important because
• There are so many different proteins with so many
  functions.
• Over 2000 different proteins/cell (humans make
  over 40K)
• So complex and sensitive to changes-cause big
  change
• Often the product of a biotech company!
• Foods/Environmental,Pharmaceuticals/Drug
  Development, Industrial Products

Check out Chapter 5 !
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Sickle Cell Anemia is caused by a faulty protein.
One nitrogen base change in DNA causes one amino
acid change in hemoglobin protein causing a huge
change in the structure and function in red blood
cells. Scientists are working on new gene
therapies to treat the cause of the abnormal
shape seen in red blood cells (left) of sickle
cell anemia patients. One DNA nucleotide mistake
causes the wrong protein shape causing the wrong
cell shape.
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Examples of Protein Function

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Why Study Proteins?

• To increase scientific knowledge
• (basic science)
• i.e. evolutionary studies
• i.e. biochemical pathways
• i.e. understand processes
• To make a (protein) product.
• i.e. Herceptin (breast cancer antibody)
• must understand structure and function to
  produce, purify and quantify proteins during
  manufacturing

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Protein Production at a Typical Biotech Co.

• Determine the new protein product to be made.
  Learn to assay for it.
• Cells are genetically engineered to make the
  new proteins.
• Engineered cells are scaled up to progressively
  large volumes, producing larger amounts of
  protein.
• Protein must be assayed.
• Protein must be purified.
• Protein must be formulated.

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Protein Manufacturing

• Protein Characterization
• Assay Development
• Protein Engineering
• Protein Purification

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How are Proteins Studied/Characterized

• Proteins are extracted from cells or media.
• Precipitated, concentrated, crystallized,
  purified
• Purified through column chromatography
• Molecular weight determined thru mass
  spectrometry.
• Size determinations and other characteristics
  through gel electrophoresis
• Amino Acid sequence determinations
• 3D structure studied through X-ray
  crystallography and computer molecular modeling
• Protein function through assay development.

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Protein Characterization

• Complicated 3-D Structure
• Must identify the AAs, their number, and their
  order to elucidate the structure and function of
  a protein.
• 4 levels of complexity
• 1 - order of AAs in chain
• 2 - folds and helices in chain due to H-bonds
• 3 - due to S-S, H-bonds, charged nonpolar
  interactions
• 4 - 2 or more chains in the protein

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Separating Proteins via SDS-PAGE Gels
(Polyacyrlamide Gel Electrophoresis)
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PAGE Gels

• On denaturing gels, can learn
• of chains in the protein.
• Size (kD) chain and approximate AA
• Size (kD) protein and approximate AA

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Visualizing Protein Samples on Gels

• Gels are stained using either
• Coomassie Blue Staining
• Silver Staining

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ASSAYS tests

• Presence - is the protein of interest there
• Activity - is the protein functional
• Concentration - how much protein is present
• Stability/Shelf-life or other characteristics

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ELISA Enzyme-Linked Immunosorbent Assay

• Bind proteins to wells in trays
• Stain it for a specific protein (using antibody
  technology)

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Western Blots

• Take a gel and transfer the proteins on it to a
  nitrocellulose or PVDF membrane
• Stain it for a specific protein (using antibody
  technology)

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Activity Assay

• Enzyme convert substrate to product
• Can measure substrate decrease or product increase

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Using the Spectrophotometer to Study Molecules
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How Protein Concentration Affects Absorbance
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Determining the Concentration of a Protein using
a Spectrophotometer
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UV Spectrophotometry

• UV/VIS Specs have 2 light sources
• UV Deuterium 240-320 nm
• White Light Tungsten 350-700 nm
• UV light is good for
• Colorless molecules
• Small amounts of sample
• UV/VIS Spec Advantages
• Can retrieve unaltered sample
• Can use tiny amounts of sample
• Most include pre-programming
• Good for concentration and
• purity testing

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Other Spectrophotometers

• Spectrophotometry
• Technology is used in
• ELISA Plate Readers
• Column Chromatography
• Genomics (gels capillary)
• FPLC
• HPLC
• Mass Spectrometry

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DNA/Protein Engineering

• Transformation of E. coli with the pAmylase
  plasmid
• Production of rAmylase

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Protein Purification

• Column Chromatography
• Gravity Flow Column
• FPLC - fast performance liquid
• HPLC - high pressure liquid

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• Biotechnology Science for the New Millennium
• Chapter 1 What is Biotechnology?
• Chapter 2 The Raw Materials of Biotechnology
• Chapter 3 The Basic Skills of the Biotechnology
  Workplace
• Chapter 4 Introduction to Studying DNA
• Chapter 5 Introduction to Studying Proteins
• Chapter 6 Finding a Potential Biotechnology
  Product
• Chapter 7 Developing Assays for Biotech Products
• Chapter 8 Modeling the Production of a
  Recombinant Product
• Chapter 9 Bring a Biotechnology Product to
  Market

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Biotechnology Science for the New Millennium
Ellyn Daugherty www.BiotechEd.com

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