Transcript Class 19

Last updated Nov. 15, 2011 5:11 PM
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Western blotting
detect the antibody use a
*Tosecondary
antibody against the
primary antibody (e.g, goat antirabbit IgG).
The secondary antibody is a
commercial fusion protein with
an enzyme activity (e.g.,
alkaline phosphatase).
*
http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html
The enzyme activity is detected
by its catalysis of a reaction
producing a luminescent
compound.
Detection of antibody binding in western blots
ECl = extended chemiluminescence
Antibody to protein on membrane
Horseradish peroxisase fusion protein, e.g.
Non-luminescent substrate
Luminescent product
(chemiluminescence)
Secondary antibody
(e.g., rabbit anti-mouse IgG)
Protein band on membrane
Luminol
Horseradish
Peroxidase (HRP)
Detect by exposing to film
(minutes or hours). Can be
quantitative.
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Western blotting
WB = western blot
Pulldown result
FLAG and Myc are epitopes for
which there are good antibodies
available.
GST = glutathione-S-transferase
PABP2 = PolyA binding protein 2
RRMs = PABP2 RNA recognition
motif
PABP2-FL full length protein
PABP2-N N-terminal fragment
Cotranfect.
Myc-SKIP = SKIP protein with a
myc tag
Ig
H-chain
Reagents
work
antiPABP2 co-IPs
SKIP
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Far western blotting to detect specific protein-protein interactions.
Use a specific purified protein as a probe instead of the primary antibody
To detect the protein probe use
an antibody against it.
Then a secondary antibody
against the first antibody, a
fusion protein with an enzyme
activity.
protein
protein
The enzyme activity is detected
by its catalysis of a reaction
producing a luminescent
compound.
OR:
Use a radioactively labeled
protein of interest and detect
by autoradiography
http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html
How to make a radioactively labeled protein:
Expression via in vitro transcription followed by in vitro translation
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T7 RNA polymerase
binding site (17-21 nt)
cDNA
VECTOR
….ACCATGG…..
Radioactively labeled protein
1. Transcription to mRNA via the T7 promoter + T7 polymerase
2. Add a translation system:
rabbit reticulocyte lysate or wheat germ lysate
Or:
E. coli lysate (combined transcription + translation, TnT)
All commercially available as kits
Add ATP, GTP, tRNAs, amino acids, label (35S-met),
May need to add RNase (Ca++-dependent, stop with EGTA) to remove endogenous
mRNA In lysate
NOTE: Protein is NOT at all pure (1000s of lysate proteins present), just ~“radio-pure”
Surface plasmon resonance (SPR)
Popular instrument is a Biacore
The binding events are monitored in real-time and it is not
necessary to label the interacting biomolecules.
In a flow cell
glass plate
Reflection angle changes depending on the mass of the material on the surface.
Binding increases this mass. Follow as a function of concentration  Kd’s
Or time : Measure on-time, off time; Kd = off-time/on-time
http://home.hccnet.nl/ja.marquart/BasicSPR/BasicSpr01.htm
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A Biacore result
Ligand
added
Ligand
removed
Back to protein-protein interactions:
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Reporter
enzyme
F = reporter protein fragment
SW Michnick web site: http://michnick.bcm.umontreal.ca/research/images/pca_general_en.gif
Enzyme fragments
themselves do not
associate well enough
to reconstitute an
active enzyme
Back to protein-protein interactions:
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Reporter
enzyme
F = reporter protein fragment
SW Michnick web site: http://michnick.bcm.umontreal.ca/research/images/pca_general_en.gif
Enzyme fragments
themselves do not
associate well enough
to reconstitute an
active enzyme
Dihydrofolate reductase (DHFR):
role in metabolism
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Folic acid
DHFR
(FH2)
DHFR
(FH4)
http://www.nature.com/onc/journal/v22/n47/images/1206946f1.gif
Clonal selection and in vivo quantitation of protein interactions with protein-fragment
complementation assays, I. Remy and S.W. Michnick PNAS 96, 394–5399, 1999
DHFR fragments
Rapamycin
promotes
the
association
of the 2
protein
domains
fMTX
Cell
growth
assay: CHO
DHFR- mutant
cells
Fluorescein – MTX
binding assay
IN PURINE-FREE MEDIUM
DHFR = dihydrofolate reductase
DHF=dihydrofolate = FH2
THF=tetrahydrofolate = FH4
fMTX=fluorescent methotrexate
FK506 = immunosuppressant drug
FKBP = FK506 binding protein
FRAP = FKBP12–rapamycin associated protein
FRB= FKBP–rapamycin binding domain of FRAP
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FK506 recruits FKBP to bind to calcineurin and
inhibit its action as a specific phosphatase
a phosphatase
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No. of CHO colonies
Claim detection of
0.05 nM rapamycin
??
[rapamycin]
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Fluorescent
methotrexate
(fMTX) assay:
Wash in, wash out
CHO cells
(permanent transfection)
cos cells
(transient transfection)
Background association of
FKBP and FRB without rapamycin
(compare mixed input)
Leucine zipper protein
fragments instead of
rapamycin binding
proteins (positive contro)
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No. of cells
Fuorescence-activated flow cytometer
(FACS is this, plus more)
Allows quantitation of fluorescence
per cell
8-fold increase in
fluorescence per cell
Fluorescence intensity
Log of fluorescence intensity
Measure affinity for a drug in vivo
[rapamycin]
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Another domain-domain interaction measured:
Erythropoietin-erythropoietin receptor (dimer) interaction: Efficacy of a peptide mimetic
EPO
EPO bp2
EPO bp1
Erytropoietin (EPO) receptor
In vivo assay of drug effectiveness (EMP1)
(inexpensive substitute for erythropoietin?)
EMP1 = Erythropoietin mimetic peptide 1
Erythropoietin
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FACS =
Fluorescence-activated
cell sorter
Impart a charge
on the recognized cell
Can be used purely
analytically
without the sorting
capability. Then
called “flow cytometry”,
or also called FACS
anyway.
Less than one cell or particle
per droplet. Thus the most
that most droplets contain is
one particle.
Charged plates attract droplets
containing a particle of the
opposite charge
Cells remain viable if
treated with care.
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Histogram-type display
No. of cells
No fluorescence (background autofluorescence)
Red stained
Usually a log scale
Having this much
fluorescence
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Scatter plot display
Amount of green fluorescence (log)
Analysis on 2 colors
One cell
You decide on
the positions of
of demarcations
Amount of red fluorescence (log)
Say, want high
reds but
low greens:
Instruct the
FACS to deflect
cells in this
quadrant only.
Collect and
grow or analyze
further.
Rice paper cont.
TPA = Tissue plasminogen activator, dissolves clots
Problem: Cleared quickly from bloodstream by liver
Bind to hepatocytes in liver via TPA’s kringle domain
Want to isolate a TPA mutant protein with less affinity for hepatocytes
Must be still enzymatically active of course.
Goal: to improve tissue plasminogen activator as a therapeutic “clot-busting” treatment
Means:
Reduce or eiminate the binding of tPA to liver cells, as this clears it from the blood
Authors here use a mammalian cells as the carrier of the DNA
and the cell surface as a display site.
Display was via a fusion protein to a membrane anchor protein, DAF (peptide, really).
DAF = “decay accelerating factor”
What did they do?
Cassette mutagenesis.
What region?
333 bp K1 (kringle-1), known to bind the MAb387, which competes for hepatocyte
binding (so assuming it is the same target epitope).
How did they get kringle mutated?
Error-prone PCR
How did they isolate just the kringle 1 region?
PCR fragment.
How did they get the mutagenized fragment back in?
Introduced restriction sites at the ends, w/o affecting the coding.
What did they put the mutagenized fragment into?
DAF – TPA fusion protein gene
How did they get it into into cells?
Electroporation
What cells did they use as hosts?
293 carrying SV40 large T antigen
How many copies per cell. And why is that important?
One, by electroporation at low DNA concentration.
[In a transient transfection!]
Binding is dominant. Lack of binding (what they are after) is recessive.
How did they select cells making MAb387-non-binding TPA?
FACS:
Recover cells that bind fluorescent mAb vs. protease domain
but low binding to fluorescent mAb vs. kringle domain
Tracked down vector: contains SV40 ori and is transfected into 293 cells making SV40
T-antigen. So plasmid replicates during the transient transfection  higher signal.
,
Sort the cells with
low fluorescence
For
reiteration
of the
process
How did they recover the plasmid carrying the mutant TPA gene
from the selected cells?
Hirt extraction: Like a plasmid prep, lyse cells gently, high MW DNA entangles and
forms a “clot”.
Centrifuge. Chromosomal DNA  soft pellet; plasmid DNA circles stay in supernatant.
Then re-transfect, re-sort in FACS.
After 2 sorting rounds, test individual E. coli clones: 60% are binding-negative.
MAb to protease domain
enriched
Collect these
No good
good
good
good
good
Log plots
Low kringle-1 reactivity
MAb to kringle-1 domain
FITC = fluorescein reagent.
PE = phycoerythrin (fluorescent protein)
Hepatoma cell binding. How?
Clone mutated regions into regular TPA gene for testing
(no DAF, protein now secreted)
Label WT TPA with fluorescein (FITC, conjugated chemically)
Mix with hepatoma cells and analyze on a flow cytometer (FACS w/o the
sorter part).
See specific and non-specific binding. Subtract non-specific binding:
the amount not competed by excess un-labeled wt TPA.
FITC = fluorescein isothiocyanate
Hepatoma cell binding assay:
measure competition for
binding of fluorescently
labeled WT TPA
Binding assay,
initial condition
Can’t compete (good)
No competitor
WT
Compete.
So still bind.
But still have
protease activity
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Got this far
Mammalian cell genetics
Introduction:
Genetics as a subject (genetic processes that go on in somatic cells:
that replicate, transmit, recombine, and express genes)
Genetics as a tool. Most useful the less you know about a process.
4 manipulations of genetics:
1- Mutation:
in vivo (chance + selection, usually); targeted gene knock-out or
alteration
in vitro: site directed or random cassette
2- Mapping:
Organismic mating segregation, recombination (e.g., transgenic mice);
Cell culture: cell fusion + segregation; radiation hybrids; FISH
3- Gene juxtaposition (complementation):
Organisms: matings  phenotypes of heterozygotes;
Cell culture: cell fusion  heterokaryons or hybrid cells
4- Gene transfer: transfection
Mammalian cell genetics
Advantages of cultured cells (vs. whole organism):
numbers, homogeneity
Disadvantages of cultured mammalian cells:
limited phenotypes
limited differentiation in culture (but some phenotypes available)
no sex (cf. yeast)
Mammalian cell lines
Most genetic manipulations use permanent lines,
for the ability to do multiple clonings
Primary, secondary cultures, passages, senescence.
Crisis, established cell lines, immortality vs. unregulated growth.
Most permanent lines = immortalized, plus "transformed“,
(plus have abnormal karyotypes)
Mutation in cultured mammalian cells:
Problem of epigenetic change: Variants vs. mutants
Variants could be due to:
Stable heritable alterations in phenotype that are not due to mutations:
heritable switches in gene regulation (we don’t yet understand this).
DNA CpG methylation, histone acetylation / de-acetylation
Diploidy. Heteroploidy. Haploidy.
The problem of diploidy and heteroploidy:
Recessive mutations (most knock outs) are masked.
(cf. e.g., yeast, or C. elegans, Dros., mice): F2  homozygotes)
Solutions to diploidy problem:
Double mutants (incl. also mutation + segregation, or mutation + homozygosis:
(rare but does occur)
Heavy mutagenesis, mutants/survivor increases but mutants/ml decreases.
How hard is it to get mutants? What are the spontaneous and induced mutation rates?
(loss of function mutants)
Spont: ~ 10-7/cell-generation
Induced: ~ 2 x 10-4 to 10-3 /cell (EMS, UV)
So double knockout could be 0.00072~ 5X10-7. One 10cm tissue culture dish holds ~
5x106 cells.
Note: Same considerations for creation of recessive tumor suppressor genes in cancer:
requires a double knockout. But there are lots of cells in a human tissue or in a mouse.
RNAi screen, should knock down both alleles: Transfect with a library of cDNA
fragments designed to cover all mRNAs. Select for knockout phenotype (may require
cleverness). Clone cells and recover RNAi to identify target gene.
A human near haploid cell strain. Use of it: Science, 326: 1231-1235 (2009)
EMS = ethyl methanesulfonate: ethylates guanine
UV (260nm): induces dimers between two adjacent pyrimidines on the same DNA strand
L
R
Homozygosis:
Loss of heterozygosity (LOH)
by mitotic recombination between
homologous chromosomes (rare)
L
R
L
M
i
t
o
s
i
s
R
--
R
L
+
-
+
2 heterozygotes again
L
R
L
R
or
+
+
-
-
Paternal
Maternal
Chr. 4, say Chr. 4
Heterozygote
+
-
+
-
Recombinant
chromatids
After homologous recombination
(not sister chromatid exchange)
Recessive phenotype is unmasked
+
+
-
1 homozygote +/+
1 homozygote -/-
= a mechanism of homozygosis of recessive tumor suppressor mutations in cancer
-
Mutagenesis (induced general mutations, not site directed)
Chemical and physical agents:
MNNG
point mutations (single base substitutions)
EMS
“
“
Bleomycin
small deletions
UV
mostly point mutations but also large deletions
Ionizing radiation (X-, gamma-rays)
large deletions, rearrangements
Dominant vs. recessive mutations;
Dom. are rare (subtle change in protein), but expression easily observed,
Recessives are easier to get (whatever KO’s the protein function), but their
expression is masked by the WT allele.
Categories of cell mutant selections
Example
purine requiring
•
Auxotrophs
•
Drug resistance
Dominant
Recessive
ouabainR, alpha-amanitinR
6TGr, BrdUr
•
Antibodies vs. surface components
MHC-
•
Visual inspection
G6PD-, Ig IP-
•
FACS = fluorescence-activated cell sorter
DHFR-
•
Brute force
IgG-, electrophoretic shifts
•
Temperature-sensitive mutants
3H-leu resistant