Protein Expression

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Protein Expression
DNA RNA Protein
1. Transcriptional Efficiency 2. Translational
Efficiency 3. Protein Stability
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Transcription Efficiency
1. mRNA Stability 2. Stem-Loop Structures
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Transcriptional Efficiency
tac Promoter System
Pribnow Box Consensus - TATAAT -35 Consensus -
TTGACA
TRP lac UV5 TTGACA
TATAAT
tac Promoter System
Features Regulation and Strength
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Translational Efficiency
1. Shine-Dalgarno Sequence 2. Codon Bias
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Constitutive Expression
Stationary Phase
Death Phase
Cell Density
Log Phase
Lag Phase
Time
Potential Advantages 1. Soluble, Folded,
Functional Protein 2. Cofactor Co-Expression
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Temperature-Inducible System
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Expression of Fusion Proteins
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Affinity Purification Handle
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His-Tag System
His6
Target
His
Fusion
His
Target
His
His
Fusion
His
Target
His
Low pH or Imidazole
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Prokaryotic vs. Eukaryotic
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Prokaryotes vs. Eukaryotes
Differences
Similarities
1. Nucleus in Euks
1. Cell Membrane
2. Organelles in Euks
2. Ribosomes
3. Intron/Exon Structure
3. DNA
4. Ribosome binding mode cap vs. RBS
5. Enhancers in Euks
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Key Expression Parameters
1. Promoters are organism specific, and tissue
specific
2. Ribosome binding site in prokaryotes
3. Codon Bias
4. Eukaryotic proteins are post-translationally
modified
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Baculovirus Structure
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Baculovirus Life Cycle
Early Phase 0-6 hrs post-infection Core DNA
enters nucleus
Middle Phase 6-20 hrs post-infection Viral
replication, nucleocapsids move back to
cytoplasm and bud-off
Late Phase gt20 hrs post-infection polh gene
expressed - huge amounts of Polyhedrin protein
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Polyhedron Gene is Non-Essential
Promoter
ATG
Stop
Target replaces Polyhedron
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Advantages of Baculovirus System

• Eukaryotic Environment for Proteins
• 1. Disulfide bonds
• 2. Post-translational modifications
• Exceptionally high expression (up to 1 g/l)
• Capacity for large insertions (100 kb)
• Virus is safe, infects limited insects

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SDS-PAGE Blotting/Hybridization
http//web.mit.edu/esgbio/www/rdna/rdna.html
http//www.uct.ac.za/microbiology/sdspage.html
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SDS PAGE
http//www.davidson.edu/academic/biology/courses/M
olbio/SDSPAGE/SDSPAGE.html
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SDS-PAGE
http//www.bergen.org/AAST/Projects/Gel/ElecProtei
ns.htm
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The Gel Apparatus
http//www.ruf.rice.edu/bioslabs/studies/sds-page
/gellab2.htmlgelprep
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Discontinuous SDS-PAGE Gels
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High molecular weight range (weights in
kilodaltons) myosin (205 kd) - from rabbit
muscle beta-galactosidase (116 kd) - from
Escherichia coli phosphorylase B (97.4
kd) - from rabbit muscle bovine serum
albumin (66 kd) egg albumin (45 kd)
carbonic anhydrase (29 kd) - from bovine
erythrocytes Low molecular weight range
bovine serum albumin egg albumin
glyceraldehyde-3-phosphate dehydrogenase (36 kd)
- from rabbit muscle carbonic anhydrase
trypsinogen (24 kd) - from bovine pancreas
soybean trypsin inhibitor (20.1 kd)
alpha-lactalbumin (14.2 kd) - from bovine milk
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Loading Quantity
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Electroblotting
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http//chem2.uscolo.edu/College/Biology/Faculty/DC
aprioglio/Classes/CellBiol/Westdectfig.html
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2D SDS-PAGE
Culture Grown Under Condition A
Culture Grown Under Condition B
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Molecular Diagnostics

• Detection of biological molecules as indicators
• of disease
• Molecular recognition - binding specificity
  allows
• identification of one molecule out of millions
• Point-of-care diagnostics
• DNA or protein-based

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Antibody Structure and Function
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Antibody Binding PocketComposed of CDR Loops
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Antibody Generation by B Cells
http//www.ultranet.com/jkimball/BiologyPages/B/B
_and_Tcells.htmlB_Cells
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Monoclonal Ab Hybridomas
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Polyclonal Antibody Production

• Immunize host animal (rabbit, goat, sheep)
• Purify antibodies from serum or use whole
  antiserum
• Contains all circulating antibodies, including
  those that bind
• to the target antigen
• POLYclonal mixture of antibodies that each
  recognize an
• epitope on the target antigen
• Both polyclonal and monoclonal antibodies can be
  produced
• in ascites by IP injection of hybridomas into
  mouse

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Antibody-Based Diagnostics

• ELISA (Enzyme-Linked ImmunoSorbant Assay)
• Sandwich ELISA
• Dipstick Assay (a type of sandwich ELISA)
• Based on antibody detection of an immobilized
  protein,
• followed by colorimetric detection
• Same principle as Western Blotting and Immuno-
• histochemistry

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Enzyme-Linked ImmunoSorbent Assay (ELISA)
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ELISA Color Development
Substrate (colorless)
Product (color)

• Secondary antibody is conjugated to an enzyme
• Enzymes alkaline phosphatase (AP), horse
  radish peroxidase (HRP)
• Substrate solution is added to the ELISA plate
• Enzyme catalyzes substrate into a colored
  product which is detected
• spectrophotometrically

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Sandwich ELISA
1) Immobilize first primary antibody to assay well
2) Add test solution containing antigen
3) Add second primary antibody
4) Add enzyme-conjugated secondary antibody
5) Add substrate and develop color
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Web Resources for ELISA Assay
http//www.hhmi.org/grants/lectures/1996/vlab/
This is a virtual ELISA lab where you can go
through the steps in detection of a
disease-related antigen.
http//www.biology.arizona.edu/immunology/activiti
es/elisa/technique.html?
This site gives an overview of the ELISA
technique, including practice problems and
animated cartoons of the assay procedure.
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ELISA Lab Experiment
Detection of streptavidin bound to biotinylated
BSA
E
E
E
b-BSA
BSA
b-BSA
BSA
BSA
BSA
b-BSA
BSA
BSA
BSA
BSA
Chilkoti et al., PNAS, 1995.
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ELISA Quantitation Interpretation
Streptavidin Biotin-BSA lt---gt Bound
Streptavidin-B-BSA
Ka SA-B-BSA/SAB-BSA
Bound
Starting Concentrations
0
Y
X
Final Concentrations
Z
X-Z
Y-Z
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Law of Mass Action
e.g. 107mM Z/1-Z1-Z when XY 1 mM
also essentially 100 bound when XY 1 nM only
sensitive when reactants near Ka This becomes a
sensitivity problem
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Mis-Interpretation of EC50 Values
To distinguish different Kas or EC50s, then
must be in correct concentration range
At Fixed Immobilized Ligand Concentrations
Bound
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Hybridization and Blotting
1. Southern Hybridization 2. Northern
Hybridization 3. Western Blotting 4. Chip and
Array Technologies
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Southern Hybridization
http//ntri.tamuk.edu/immunology/abproduction2.htm
l
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Screening Oligo or Antibody
Purify Target Protein
Genome Sequence
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Gen Bank
InCyte
Gene Sequences Clones
Order gene in plasmid
Receive clone in mail
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Northern Hybridization
Isolate mRNA (Different Lengths)
Probe with labeled DNA
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Chromosomal DNA Analysis
http//science-education.nih.gov/newsnapshots/TOC_
Chips/Chips_RITN/How_Chips_Work_1/how_chips_work_1
.html
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Conventional DNA Hybridization
Using a single probe. In the 1970s, scientists
learned to use DNA probes to find specific
target sequences in solution. First,
radioactively label a known DNA sequence, then
put it into a mix of unknown sequences. If the
probes complement is there, it will bind.
Look for the label. Next they separated the
double-stranded DNA from the single-stranded. If
the probe found its target, the radioactive
label would be in the double stranded fraction
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DNA Chip Approach
DNA Chips Thousands of
Probes at Once. DNA chips
allow scientist to use thousands of probes all at
once. First, spot the
different probes on a surface, noting sequence
they put at each spot.
Let The Targets Loose. This time, label the
targets in solution and put the solution on the
chip. Any targets that find their complementary
probes will stick to the surface.
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Readout
Look For The Label. Next,
gently wash the surface, and
look for the labeled spots. Because you know the
sequences of all the probes,
you can easily deduce the sequences present
in the solution.
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DNA Hybridization on Chips
Nanogen Technology
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Compare gene expression under two conditions over
whole genome
http//www.cs.wustl.edu/jbuhler//research/array/
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The Scientific Journal Article

• 1. Title
• Express Most Important Concept
• 3rd Person, Active Tense
• Brief and Catchy

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The Scientific Journal Article

• 1. Abstract
• Miniature Version of Intro, Methods, Results,
  Significance
• Self-Contained Without Reference to Text
• I Prefer 3rd Person Exclusively in Abstract
• No Footnotes or Abbreviations
• Literature References Must be Complete
  Citations

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The Scientific Journal Article

• 3. Introduction
• States the Purpose of the Current Study
• States the Current Deficiencies in Knowledge
• Directly Relevant Background Only (not review)
• Remember to Acknowledge Potential Reviewers

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The Scientific Journal Article

• 4. Materials and Methods
• Theoretically Sufficient for Others to Repeat
  Work
• Reality Is That You Wont Find That Much Info
• Do Not Detail Previously Published Procedures -
  
• Reference Previous Papers
• Break Into Subheadings, e.g. Chemicals,
  Site-Directed Mutagenesis, Protein Expression
• Theoretically Have To Make All Constructs
  Available

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The Scientific Journal Article

• 5. Results
• Third Person, Stick to the Facts
• Tables and Figures Should Not Overlap in
  Content
• Think Long and Hard About Figure Content
• Supplementary Data Can Be Appended

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The Scientific Journal Article

• 6. Discussion
• What did you learn and how does it fit into
  existing picture
• Draw together results and show
  inter-relationships
• You are allowed to speculate a bit, but not too
  much
• Avoid literature reviews but you can pat a few
  backs, or
• point to discrepencies
• This is a chance to claim territory

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The Scientific Journal Article

• 7. Acknowledgements
• Material gifts, advice, technical assistance,
• secretarial assistance
• Funding agencies
• Avoid thanking the academy and your first grade
  teacher

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The Scientific Journal Article

• 8. References
• Must format to journal style
• Only articles accepted can be cited in
  references - others
• go as unpublished results or personal
  communication

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The Scientific Journal Article

• 9. Figures and Tables
• Figure legends must have symbols, and state
  result succinctly
• Figures need to be perfect
• Tables formated to journal specifications

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The Scientific Journal Article

• Communications vs. Articles
• 1. Communications
• Exciting findings, high significance in hot
  field
• Typically shorter, often without complete
  story, single concept
• Usually imply a full article is forthcoming
• Reviews are accept or reject without
  significant revisions
• Compress Results and Discussion, minimize
  methodology
• 2. Articles
• Full length, full examination of problem
• More experimental detail
• Reviewers often suggest important revisions,
  which you
• can respond to

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The Scientific Journal Article

• Review of Articles
• Generally 2-3 reviewers
• Options are Accept w/o Revisions, Accept w/
  Minor
• Revisions, Accept only after Major Revision
  (usually
• reviewed again), or Reject
• Authors can suggest suitable reviewers and
  disqualify
• others dont review

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Journal Assignment
1. Write an Introduction to a paper that would
report the site-directed mutagenesis and
mutant production that you have performed in
class. This would cover your work through
the production of the mutant. 2. Write a
Results section to the same paper that covers
your key results such as plasmid isolation
and restriction mapping, site-directed
mutagenesis and sequencing, expression, and
SDS-PAGE/ELISA protein characterization. Create
Figures corresponding to key results.
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Introduction to Molecular Cloning
I. What is Cloning II. Cloning Strategies A.
Classical B. PCR III. Example A.
Antibody Engineering
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How Do You Get a Gene?
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Total Gene Synthesis
Gene Built from Oligonucleotides
Gives Choice of DNA Sequence Optimize
Expression Optimize Cut Site Placements Easier
Site-directed Mutagenesis
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What is Cloning
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Genomic Libraries
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cDNA Libraries
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Expression Libraries
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Screening for Target Sequence
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Screening Oligonucleotides
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Screening Oligo or Antibody
Purify Target Protein
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Screen Expression Library
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PCR Cloning
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PCR Cloning
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Web-Site For Review
http//esg-www.mit.edu8001/esgbio/rdna/rdna.html