Vector religation control

1
Vector re-ligation control
If you are cloning into a vector that does not
have a lacz gene, what could you do to estimate
how much of your vector re-ligates?
What are possible causes of vector re-ligation?
2
Vector re-ligation control

digested insert
digested plasmid
digested plasmid
Ligate and transform
Ligate and transform
3
Shotgun vs. Directed cloning
Clone of interest
4
Lecture objectives Cloning and PCR Compare and
contrast shotgun and directional
cloning Identify the most appropriate cloning
method for a given experiment Recognize the
components of a plasmid Differentiate between
selection and screening Understand how the
polymerase chain reaction works and how it
relates to biological processes Design PCR
primers for a given template and develop a simple
cloning strategy
5
Protein Expression Systems

• Biochemistry 651
• March 25, 2009

6
Lecture objectives Protein expression systems
Identify key features of 4 different expression
systems 1) Bacterial 2) Insect cell 3)
Mammalian 4) Cell Free
Identify the promoter for each expression system
Compare the advantages and limitations of the
various systems
Select an expression system for a given
experimental situation and be able to explain
why you chose it.
7
Protein expression overview
1. What are you going to express and why?
2. Read the literature
3. Decide on your system prokaryotic,
eukaryotic, cell free

• Subclone your gene into the system (you may need
  to use
• PCR).

5. Introduce the gene into cells by
transformation, transfection, or infection.
6. Do a time course to determine when the
protein is expressed.
8
Our friends Recombinant proteins

• Recombinant Human Insulin synthesized in E.
  Coli
• Previously insulin was purified from bovine or
  porcine tissue

• Tissue Plasminogen Activator (tPA) an enzyme
• synthesized in a eukaryotic cell line (CHO cells)
• Used to dissolve blood clots in patients
  suffering from
• heart attacks and strokes

9
Transcription and translation
http//www.youtube.com/watch?vbk7PW1FKMTI
Recombinant protein expression takes advantage of
the cells machinery to make our protein of
interest. We provide the gene and a good
promoter and the cell does the rest.
10
Bacterial Expression System
Target gene
Clone your gene into the appropriate cloning
vector for protein expression
Target gene
Novagen pET System Manual 11th Edition January
2006
11
Bacterial Expression System
Your gene must be cloned in-frame with the start
codon.
pET vector multiple cloning region
T7 terminator sequence
T7 promoter sequence
ATGGGCAGCCATCATCATCATCATCATAGCAGCGGATCC
His-tag
start codon
BamHI
Clone your gene into the BamHI site
ATG GGC AGC CATCATCATCATCATCAT AGC AGC GGA TCC
ATG GATCC
start codon
His-tag
Your genes start codon
Transform your clone into the appropriate
bacterial host
Novagen, pET System Manual 11th Edition, January
2006
12
Bacterial Expression System
Target gene is under control of theT7 promoter
T7 RNA polymerase is not naturally expressed
in E. Coli
Therefore, you must use host cells that
express T7 RNA polymerase
Novagen pET System Manual 11th Edition January
2006
13
Bacterial Expression System
Once you add IPTG to induce expression, you do a
time course to determine the time of maximum
protein expression.
Remove aliquots at various time points
Add IPTG
SDS-PAGE
corbis
corbis
analyze aliquots
culture of bacteria containing recombinant plasmid
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Bacterial Expression System

• ADVANTAGES
• Relatively fast (1-2 weeks including cloning)
• Inexpensive
• High expression levels
• System is amenable to large scale preparations

• DISADVANTAGES
• Protein may be insoluble
• Minimal post-translational modifications (ie.
  glycosylation,
• phosphorylation, disulfide bonds)
• 3) Protein may be mis-folded
• 4) Expression of functional mammalian proteins
  may be
• difficult

15
Eukaryotic cell Expression System
Viruses can be used to get your gene into a
eukaryotic cell.
Isolate viral genome
Insert your gene
For our purposes, a virus is a protein shell
with a nucleic acid genome inside.
put recombinant viral genome into cells
Cells make a lot of recombinant virus
(not even remotely to scale)
Infect cells with recombinant virus
Your Protein (ta-da)
16
Insect cell Expression System
Baculovirus is used to express the protein of
interest in insect cells.
Gene of interest is cloned under the control of a
baculovirus- specific strong promoter.
The recombinant baculovirus is created and then
used to infect insect cells.
During the course of its natural life cycle the
virus produces your favorite protein.
17
Insect cell Expression System
Clone your gene into the donor Plasmid
transposition
transformation
Bacmid baculovirus genomic DNA
Invitrogen User Manual for the Bac-to-Bac
Expression System Version A, 15 December 2008
18
Insect cell Expression System
Transfect Bacmid into insect cells
Isolate Bacmid DNA
Insect cells produce recombinant baculovirus
Bacmid gt135kb screen by PCR Not restriction
digest
Invitrogen User Manual for the Bac-to-Bac
Expression System Version A, 15 December 2008
19
Insect cell Expression System
Infect a new plate of insect cells with
the recombinant baculovirus
During the viral life cycle your protein will be
produced
Your Protein (ta-da)
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Insect cell Expression System

• ADVANTAGES
• Reasonably high expression levels
• Supports many post translational
• modifications (PTMs)
• 3) Proteins are likely to fold properly

• DISADVANTAGES
• Takes a long time (2-4 weeks to get recombinant
  virus)
• Expensive (tissue culture, transfection
  reagents, special
• host E. coli)
• 3) Complicated cloning
• 4) Must keep passaging cell lines

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Mammalian cell Expression System
Two ways to express protein in mammalian cells

• Transfection Nucleic acid can be introduced
  directly
• into the cells.

protein
Promegas Tranfection Guide Chapter 1
22
Mammalian cell Expression System
Two ways to express protein in mammalian cells
2) Cells can be infected with a recombinant
Adenovirus containing your gene of interest.
Your gene of interested is cloned downstream
of a strong, constitutive, viral
promoter. Adenovirus can infect many different
mammalian cell types (including skin, muscle,
brain, bone, nerve, and liver cells as well as
embryonic stem cells) In the course of its life
cycle the virus uses the cellular machinery to
produce your protein.
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Mammalian cell Expression System
recombination
r
36,000 bp
5000 bp
Your gene is recombined into the adenovirus
genome
Gene of interest
r
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Mammalian cell Expression System
Multiple copies of the recombinant adenovirus
genome can be made in bacteria.
1) Isolate adenovirus genome from bacteria
2) Transfect the adenovirus genome into
mammalian cells
Mammalian cells produce recombinant adenovirus
25
Mammalian cell Expression System
Infect a new plate of mammalian cells with
the recombinant adenovirus
During the viral life cycle your protein will be
produced
Your Protein (ta-da)
26
Mammalian cell Expression System

• ADVANTAGES
• Highest level of PTMs
• Best system to obtain functional human proteins

• DISADVANTAGES
• 1) Low level of protein expression
• 2) Takes a long time (2-3 weeks to get
  recombinant virus)
• Expensive (tissue culture, transfection
  reagents, special vector system)
• 4) Complicated cloning and virus generation
  system
• 5) Must keep passaging cell lines

27
Cell - Free Expression System

• 3 systems
• Rabbit Reticulocyte Lysate lysed immature red
  blood cells,
• microccocal nuclease treated to degrade
  endogenous mRNA.
• You add the RNA template.
• E. coli E. coli extract that contains T7
  polymerase.
• You supply circular DNA with your gene cloned
  downstream
• of the T7 promoter.
• 3) Wheat Germ microccocal nuclease treated
  wheat germ
• extract containing the components necessary for
  translation.
• You add the RNA template.

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Cell - Free Expression System
Amino acids
Label if needed
template
Cell-free system
salt
Incubate 60-90 min.
Analyze your protein
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cell-Free Expression System

• ADVANTAGES
• Very fast (hours vs. days or weeks)
• Easy labeling
• Little endogenous protein produced
• Flexible template DNA or RNA
• Can express toxic proteins

DISADVANTAGES 1) Very low level of protein
expression nanogram amounts 2) Very
expensive 3) Little PTM you can add reagents
to get some PTMs.
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Lecture objectives Protein expression systems
Identify key features of 4 different expression
systems 1) Bacterial 2) Insect cell 3)
Mammalian 4) Cell Free
Identify the promoter for each expression system
Compare the advantages and limitations of the
various systems
Select an expression system for a given
experimental situation and be able to explain
why you chose it.
31
In-class problem
What expression system would you use to produce
ß-galactosidase in order to determine its
structure by NMR? Why?
What expression system would you use to produce a
mutant form of tissue plasminogen activator if
you wanted to look at its function in brain and
liver cells? Why?