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Recombinant DNA II
Andy Howard
Introductory Biochemistry
20 November 2008
Biochem: Recombinant DNA
11/20/2008
Recombinant DNA (review)
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Much of our current understanding of
molecular biology, and of the ways we
can use it in medicine, agriculture, and
basic biology, is derived from the kinds of
genetic manipulations that we describe
as recombinant DNA
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What we’ll discuss
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Expression
Genomics
Proteomics
Amplification
Polymerase
Chain Reaction
Biochem: Recombinant DNA
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Mutagenesis
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Random
Site-directed
Applications
Probing proteinprotein interactions
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Using expression vectors
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We often want to do something with
cloned inserts in expression vectors, viz.
make RNA or even protein from it
RNA: stick an efficient promoter next to
the cloning site; vector DNA transcribed
in vitro using SP6 RNA polymerase
This can be used as a way of making
radiolabeled RNA
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Protein expression
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Making (eukaryotic) proteins in bacteria via
cDNA means we don’t have to worry about
introns
Expression vector must have signals for
transcription and translation
Sequence must start with AUG and include a
ribosome binding site
Strong promoters can coax the bug into
expressing 30% of E.coli’s protein output to be
the one protein we want!
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QuickTime™ and a
decompressor
are needed to see this picture.
Example: ptac
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This is a fusion of lac promoter (lactose
metabolism) with trp promoter
(tryptophan biosynthesis)
Promoter doesn’t get turned on until an
inducer (isopropyl--thiogalactoside,
IPTG) is introduced
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Eukaryotic expression
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Sometimes we need the glycosylations and
other PTMs that eukaryotic expression enables
This is considerably more complex
Common approach is to use vectors derived
from viruses and having the vector infect cells
derived from the virus’s host
Example: baculovirus, infecting lepidopteran
cells; gene cloned just beyond promoter for
polyhedrin, which makes the viral capsid protein
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Screening libraries
with antibodies
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Often we have antibodies that react with a
protein of interest
If we set up a DNA library and introduce it into
host bacteria as in colony hybridization, we can
put nylon membranes on the plates to get
replicas of the colonies
Replicas are incubated to make protein
Cells are treated to release the protein so it
binds to the nylon membrane
If the antibody sticks to the nylon, we have a hit!
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Fusion proteins
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Sometimes it helps to co-express our protein of
interest with something that helps expression,
secretion, or behavior
We thereby make chimeric proteins, carrying
both functionalities
We have to be careful to keep the genes in
phase with one another!
Often the linker includes a sequence that is
readily cleaved by a commercial protease
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Fusion systems (table 12.2+)
Product
Origin
Mass, Secre Affinity Ligand
kDa
ted?
-galactosidase
E.coli
116
No
APTG
Protein A
Staph.
31
Yes
IgG
Chloramphenicol
acetyltransferase
E.coli
24
Yes
Chloramphenicol
Streptavidin
Strep.
13
Yes
Biotin
Glut-S-tranferase
E.coli
26
No
Glutathione
Maltose Bind.Prot. E.coli
40
Yes
Starch
Hemoglobin
16/32
No
None
Vitreoscilla
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Improving purification
via expression
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If we attach (usually at the N-terminal end) a histag (several his, several cys) to our protein, it
becomes easier to purify:
The his tag forms a loop that will bind strongly to
a divalent cation like Ni2+
Thus we can pour our expressed protein
through a Ni2+ affinity column and it will stick,
while other proteins pass through
We elute it off by pouring through imidazole,
which completes for the Ni2+ and lets our protein
fall off
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Protein-protein interactions
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One of the key changes in biochemistry over
the last two decades is augmentation of the
traditional reductionist approach with a more
emergent approach, where interactions
among components take precedence over
the properties of individual components
Protein-protein interaction studies are the
key example of this less determinedly
reductionist approach
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Two-hybrid screens
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Use one protein as bait; screen many
candidate proteins to see which one
produces a productive interaction with
that one
Thousands of partnering relationships
have been discovered this way
Some of the results are clearly
biologically relevant; others less so
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2-hybrid
screen
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X is bait, fused
to DNA binding
domain of GAL4
Y is target, fused
to transcriptional
activator portion
of GAL4
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Reporter constructs:
How to study regulation
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Put a regulatory sequence into a plasmid
upstream of a reporter gene whose product is
easy to measure and visualize
Then as we vary conditions, we can see how
much of the reporter gets transcribed
Example: Green Fluorescent Protein, which can
be readily quantified based on fluorescent yield
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Genomics
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Application of these high-throughput
techniques to identification of genetic
makeup of entire organisms
First virus was completely sequenced
in the late 1970’s
First bacterium: Haemophilus
influenzae, 1995
Now > 50 organisms in every readily
available phylum
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What’s been
sequenced?
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Current list
would be
even longer
Also include
multiple
individuals
within a
species
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How genomics works
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A researcher who wishes to draw general
conclusions about structure-function
relationships may want to learn the sequence
(“primary structure”) of many genes and nongenomic DNA in order to draw sweeping
conclusions or build a library of genetic
constructs, some of which he will understand
and others he won’t
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Complete sequencing of a
genome
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Fragment chromosomes
Shotgun sequencing of fragments
Reconstitution based on overlaps
Cross-checking to compensate for errors
Interpretation
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Human genome project
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Effort began in late 1980’s to do complete
sequencing of the human genome
Methods development was proceeding
rapidly during the period in question so it
“finished” well ahead of schedule in 1999
Partly federal, partly private
Related efforts in other countries
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What’s the point?
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Better understanding of both coding and
non-coding regions of chromosomes
Identification of specific human genes
Medically significant results
Statistical results (x% are Zn fingers…)
Variability within Homo sapiens or some
other sequenced organism by comparing
complete sequences or ESTs between
individuals
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Proteomics
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Analysis of the resulting list of expressible (not
necessarily expressed!) proteins
Often focuses on changes in expression that
arise from changes in environmental conditions
or stresses
Often useful to analyze mRNAs along with
proteins
Mass spectrometry is a key tool in proteomics
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How MS
works in
proteomics
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QuickTime™ and a
decompressor
are needed to see this picture.
Cartoon from
Science Creative
Quarterly at
U.British
Columbia, 2008
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Amplification
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Prokaryotic and eukaryotic cells can,
through mitosis, serve as factories to
make many copies (> 106 in some cases)
of a moderately complex segment of
DNA—provided that that segment can be
incorporated into a chromosome or a
plasmid
This is amplification
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Polymerase chain reaction
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This is a biochemical tool that enables
incorporation of desired genetic material into a
cell’s reproductive cycle in order to amplify it
Start with denatured DNA containing a segment
of of interest
Include two primers, one for each end of the
targeted sequence
The sequence of events is now well-defined after
three decades of refinement of the approach
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PCR: the procedure
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Heat to denature cellular dsDNA and
separate the strands
Add the primers (ssDNA) and
polymerase
Heat again, then cool enough for ligation
Continue cycling to get many cell
divisions ~ 106-fold amplification
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PCR in
practice
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RT-PCR
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Variant on ordinary
PCR: starting point is
an RNA probe that
can serve as a
template for DNA via
reverse transcriptase
Once cDNA copy is
available, normal
PCR dynamics apply
QuickTime™ and a
decompressor
are needed to see this picture.
Cartoon courtesy Cellular &
Molecular Biology group at
ncvs.org
Biochem: Recombinant DNA
11/20/2008
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Mutagenesis
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Procedure through which mutations are
introduced into genomic DNA
May be used:
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To generate diversity
To probe the essentiality of specific genes
To examine particular segments of genes
To alter properties of DNA or its mRNA transcript
or a translated protein
To provide information and material for gene
therapy
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Random mutagenesis
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DNA (often locally ssDNA) is exposed to
mutagens in order to introduce random
mispairings or increase the rate of
mispairing during replication
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Can involve ionizing radiation
Can involve chemical mutagens:
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Error-prone PCR
Using “mutator strains”
Insertion mutagenesis
Ethyl methanesulfonate
Nitrous acid and other nitroso compounds
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Site-directed mutagenesis
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Specific loci in DNA targeted for alteration
Typically involves excision, addition of altered
bases, and religation
Can be accomplished even in eukaryotic cell
systems
Many biochemical systems can be
systematically probed this way:
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To find essential amino acids in expressible
proteins
To see which amino acids are important
structurally
To examine changes at RNA level
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How do we use these tools?
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Already discussed significance of
complete sequencing efforts
Generally: amplification and expression
give us access to and control of
biochemical systems that otherwise have
to be isolated in their original setting
These methods enable controlled
experiments on complex systems
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Gene therapy
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Cloned variant of deficient gene is inserted into
human cells
Can be done via viral or other vector carrying an
expression cassette
Maloney murine leukemia virus works for
cassettes up to 9kbp; depends on integrating
the cassette into the patient’s DNA
Adenovirus works up to 7.5 kb: never gets
incorporated into host, but simply replicates
along with host
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Retroviral
approach
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Adenoviral
approach
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