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Proteins and Products

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Chapter 4 Proteins and Products Search for new and novel proteins Going to extremes Tropical rain forests Yellowstone hot springs and soils Branches and pine ... – PowerPoint PPT presentation

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Title: Proteins and Products


1
Chapter 4
  • Proteins and Products

2
Search for new and novel proteins
  • Going to extremes
  • Tropical rain forests
  • Yellowstone hot springs and soils
  • Branches and pine needles in hot springs are a
    potential source of thermophilic lignin-degrading
    microbes and their enzymes
  • These enzymes have commercial value in pulp and
    paper industry

3
Classes of Proteins
  • Enzymes
  • Depolymerization
  • Enzymes that degrade large complex molecules such
    as lignin, starch and cellulose to smaller
    subunit compounds
  • Amylase acts on starch to produce glucose
  • What is the degradation product of cellulose?
  • Lipases
  • Proteases
  • Hormones (chemical messengers)
  • insulin

4
Examples of proteins used in other commercial
applications
  • Textile industry
  • Enzymes have replaced toxic chemical
  • stone-washed denim
  • Home products industry
  • Enzymes added to Drano and Liquid Plumber
    replaces caustic chemicals to dissolve hair
    fibers, grease, etc. that plug drains

5
Adhesives
  • Mussel adhesive protein
  • Underwater glue
  • Industries like 3M are trying to design and
    synthesize proteins in E. coli bacterium that
    mimic the natural proteins excreted by mussels

6
Protein structures
  • Proteins are linear chains of amino acids linked
    together by a peptide bond
  • Primary structure refers to the sequence of the
    different amino acids in the chain

-aa1- aa2- aa3- aa4-
H
aa1 -C-O-N-aa2-
O
7
Protein structure
  • Secondary structure refers to the shape of the
    amino acid chain
  • Beta sheets (ribbon)
  • Alpha helix (cork screw)
  • Tertiary structure refers to the shape the chain
    takes as a result of attractions between alpha
    helices and beta sheets in different sections of
    the chain

8
Protein structure
Chain 1
  • Quaternary structure is a protein that contains
    more than one amino acid chain
  • hemoglobin

Chain 2
9
Protein folding
  • Folding of the peptide chain(s) confers structure
    to the protein
  • Certain amino acids (aa) placed at specific
    intervals along the chain established how chain
    folds
  • alpha helix
  • beta sheet
  • proline aa inserts a kink in the chain

10
Protein folding
  • Structure controls protein function
  • tangled plaques of Alzheimers disease may be
    the result in errors in protein folding
  • Some proteins have sugar residues attached to the
    peptide chain
  • glycosylation
  • affects activity of protein
  • found in eucaryotic cells

glycosylation
11
Diseases related to incorrectly folded proteins
  • Cystic fibrosis
  • mad cow disease (bovine spongiform encephalitis)
  • A substitution of one amino acid for another
    along the peptide chain can affect how the
    protein folds.

12
Bioprocessing
  • There are numerous steps between collecting raw
    material from nature and producing a commercial
    product

13
Large-scale cultivation of microorganisms
  • Fermentation vessels or bioreactors has impeller
    that stirs the culture so bacteria in suspension
    get good access to dissolved nutrients and oxygen
    for growth.

14
Food processing proteins
15
Proteins in medical applications
  • Insulin
  • Recombinant insulin produced in E. coli avoids
    adverse reactions by our immune system in
    response to foreign substances (insulin recovered
    from pigs and cattle).
  • Pharmaceutical industry has invested in this area
    because they can charge so much for use in
    treatments

16
Recombinant proteins produced by the
pharmaceutical industry
Application
Protein
  • Erythropoietins
  • Interleukin 1,2,3,4
  • Human growth factor
  • Tissue plasminogen factor
  • vaccines
  • Treatment of anemia
  • Cancer, AIDS
  • Treatment of growth deficiency in children
  • Heart attack and stroke treatment
  • Hepatitis B, herpes, malaria

17
Recombinant proteins produced in eucaryotic fungi
Fungi are good hosts for producing glycosylated
proteins since, as eucaryotic cells, they have
the molecular machinery to add sugars molecules
to peptide chains, whereas, bacteria do not have
this machinery
18
Protein engineering
  • Shotgun approach
  • induce mutations randomly into genes and then
    select organisms (bacteria) with protein products
    (enzyme) that, when assayed using an enzyme
    assay, gives the highest activity.
  • Led to recovery of an enzyme that functioned in
    presence of high cyanide concentration.

19
Protein engineering
  • Directed molecular evolution
  • Induce mutation in a specific gene and select
    best protein from mutant gene for our application
    irrespective of its value to organism. (example
    insulin production in E. coli)

20
Protein engineering
  • Hybrid proteins
  • a piece of one protein linked to a piece of
    another protein in the same amino acid chain.
  • This is often done to encourage E. coli cells to
    secrete protein into surrounding medium instead
    of holding it inside cell.
  • This makes it easier to recover and purify the
    protein.
  • An enzyme can be added after purification to cut
    off the part of the hybrid that signaled
    exportation.

signal sequence (only used to direct protein for
export out of cell
export
functional protein
protease cleavage
21
Upstream processing
Protein expression (transcription translation)
Protein export
22
Processes used to recover proteins produced by
microbes
whole culture
inside cell
outside cell
separate
centrifugation and cell lysis
proteins from
smaller molecules
dialysis
ion exchange
chromatography
23
Downstream processing
  • Step 1-Recovery of protein from cells

Cell pellet
24
Cell lysis(when protein is still inside cell)
High-energy sound waves, High salt concentration
25
Downstream processing
  • Step 2-Separation of protein from other culture
    medium components (purification)
  • sometimes this is very time-consuming and costly
  • purification is necessary because other medium
    components may interfere with protein function
    or cause adverse reaction if protein is
    introduced into a human

26
Processes used to recover proteins produced by
microbes
whole culture
inside cell
outside cell
separate
centrifugation and cell lysis
proteins from
smaller molecules
dialysis
ion exchange
chromatography
27
Processes used to recover proteins produced by
microbes
whole culture
inside cell
outside cell
separate
centrifugation and cell lysis
proteins from
smaller molecules
dialysis
ion exchange
chromatography
28
Dialysis
29
Processes used to recover proteins produced by
microbes
whole culture
inside cell
outside cell
separate
centrifugation and cell lysis
proteins from
smaller molecules
chromatography
dialysis
30
Chromatography
Separates protein of interest from other proteins
and medium components
31
Chromatography
Size-exclusion chromatography
Column filled with beads
detector
32
Chromatography
Ion-exchange chromatography
33
Identification of proteins separated in different
fractions
wells
  • SDS-PAGE
  • Sodium Dodecyl Sulfate Polyacrylamide Gel
    Electrophoresis

Works similar to agarose gel electrophoresis to
separate DNA and RNA fragments Used to following
process of separating protein of interest from
all other proteins
Demonstrates that you have a purified protein
34
Post-purification analysis
  • Protein sequencing

Go to Quick-time movie
35
Post-purification analysis
  • Protein sequencing

Go to Quick-time movie
36
Post-purification analysis
  • Protein sequencing

Go to Quick-time movie
37
Post-purification analysis
  • Protein sequencing

Go to Quick-time movie
38
Post-purification analysis
  • Protein sequencing

unknown amino acid
Go to Quick-time movie
39
Post-purification analysis
  • X-ray crystallography

40
Proteomics
  • Proteomics refers to the evaluation of the
    complement of proteins expressed by a cell under
    a certain set of conditions
  • Proteomes are compared under healthy and diseased
    states
  • Variations in protein expression are correlated
    to onset or progression of a specific disease
  • Proteomics applies electrophoresis, mass
    spectrometry and amino acid sequencing

41
Proteomics
Protein chips
Used to detect presence of specific proteins
10,000 probes
42
Pathway Tools overview diagram
43
(No Transcript)
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