Aplikovaná chemie a biochemie
Přednáška č. 2
Proteinové techniky
(1)
Proteinové techniky (1):
ˇlokalizace v buňce (live cell imaging;
imunofluorescenční techniky; frakcionace);
* interakce mezi proteiny (imunoprecipitace;
GST-pull-down; yeast two-hybrid screen);
* interakce s DNA ; detekce transkripční
aktivity
Lokalizace proteinu v buňce
* imunodetekce ­ imunofluorescenční a
imunohistochemické techniky; kolokalizace proteinů
­ konfokální mikroskopie;
Marker dané organely (subcel. komp.), negativní
kontrola.
Enzymové (HRP, AP) immunofluorescenční.
techniky. Hlavní výhody jsou senzitivita možnost
vícenásobného barvení, nevýhodou je fading,
autofluorescence a náročné technické vybavení.
Nevýhodou imunohistochemických technik je také
vznik artefaktů v důsledku fixace buněk.
AhR ve varlatech potkana:
sekundární protilátka značená
+ TCDD;
30 min
Příklad: Lokalizace Ah receptoru v buňce
AhR ve buněčné linii WB-F344:
sekundární protilátka značená FITC
Lokalizace v buňce
* live cell imaging ­ využití proteinů značených
např. fluorescenčními proteiny ­ time-lapse video,
FLIP, FRET;
Výhoda ­ možnost pracovat s živými buňkami ­ je
možné sledovat translokace proteinů, pohyby
organel, pohyb proteinů v rámci buňky, stanovit
přibližné relativní množství proteinu i proteinové
interakce (intermolecular FRET).
Fluorescenční markery:
GFP
Fluorescence can be used to visualize
cell structures at many levels.
Originally, fluorescence was mainly
observed from small organic dyes
attached by means of antibodies to the
protein of interest. However, antibody
targeting of intracellular proteins
normally requires cell fixation and
permeabilization.
Later, fluorophores could directly
recognize organelles, nucleic acids, and
certain important ions in living cells. In
the past decade, fluorescent proteins
have enabled noninvasive imaging in
living cells and organisms of reporter
gene expression, protein trafficking,
and many dynamic biochemical signals.
Fluorescenční markery:
Fluorescenční markery:
Fluorescenční markery:
Lokalizace v buňce
* subcelulární frakcionace následovaná WB
Výhoda ­ nižší spotřeba protilátek, snadné
vyloučení nespecifických vazeb protilátek;
nevýhoda ­ pracnost.
Nejčastěji používaná metoda ­ použití
specifických detergentů, ultracentrifugace.
Sequential centrifugation:
Nuclear fraction - at 1,000g for 10 min.
Heavy mitochondrial fraction 3,000g for 10min.
Light mitochondrial fraction at 15,000-17,000g for 10 min.
Microsomal fraction at 100,000g for 45 min.
Composition of the pellets
The Nuclear Pellet contains, in addition to nuclei, mitochondria, sheets of plasma
membrane (if present) and, if the homogenate has not been filtered, unbroken
cells and debris (including connective tissue). Formation of this pellet is
sometimes carried out at 500g rather than 1000g.
The Heavy Mitochondrial Pellet contains predominantly, mitochondria with rathe
few contaminants and is a common source of these organelles for respiratory
studies. Minor components such as lysosomes, peroxisomes, Golgi membranes and
various membrane vesicles are present largely because of entrapment during the
pelleting process. Some plasma membrane fragments may also be present. These
contaminants can be reduced by repeated washing.
The Light Mitochondrial Pellet contains mitochondria, lysosomes, peroxisomes,
Golgi membranes and some endoplasmic reticulum. Of all differential
centrifugation fractions it is the most variable in terms of the actual
centrifugation parameters used: g-forces of 15-20,000g and times of 10-20 min
are the most common. Some methods are designed to maintain the Golgi
membranes in their "stacked" form so that they sediment at much lower g-forces
(see ref 3 for more information)
The Microsomal Pellet is rather better defined and contains only membrane
vesicles. Some of those vesicles will have been present in the cell before
homogenization (e.g. endosomes, secretory vesicles and vesicles from the transGolgi
network), others from the plasma membrane, Golgi and smooth and rough
endoplasmic reticulum, will have been produced by the homogenization procedure.
Characterization of DR depletion
in Hepa1c1c7 cells. A,
TCDD (10 nM) was added to
Hepa1c1c7 cells for the times
indicated before whole cell protein
extracts were separated using 8%
SDS-PAGE and analyzed by
immunoblotting with anti-DR mAb.
B, TCDD (10 nM) and/or Me2SO
vehicle were added to Hepa1c1c7
cells for 2.5 h. MG132 (2.5 mM),
E64 (10 mM), or vehicle (DMSO)
were added at the same time
in the lanes shown. Cytosolic and
nuclear extracts were prepared
and analyzed by 8% SDS-PAGE and
immunoblotting using anti-DR mAb.
Interakce mezi proteiny
* stabilní ­ proteiny vytvářejí komplex
* tranzientní ­ např. interakce enzymu s
proteinovým substrátem
* afinita dvou proteinů je dána rovnovážnou
disociační konstantou ­ Kd = [A]*[B]/[AB]
Základní metody:
* GST pull-down assay
* two-hybrid methods
* co-immunoprecipitation
* FRET
GST pull-down assay
is a simple technique to test interaction between a tagged protein or
the bait (GST, His6, biotin ...) and another protein (test protein, or
prey).
The bait protein, purified from an appropriate expression system
(e.g., Escherichia coli), is immobilized on a glutathione affinity gel. The
bait serves as the secondary affinity support for identifying new
protein partners or for confirming a previously suspected protein
partner to the bait.
Prey protein can be obtained from multiple sources including
recombinant purified proteins, cell lysate or in vitro
transcription/translation reactions.
Protein-protein interactions can be visualized by SDS-PAGE and
associated detection methods depending on the sensitivity
requirements of the interacting proteins. These methods include
Coomassie or silver staining, Western blotting and [35S] radioisotopic
detection.
GST pull-down assay
Two-hybrid assays (interaction trap):
Two chimeras, one containing the
DNA-binding domain and one that
contains an activation domain, are cotransfected
into an appropriate host
strain. If the fusion partners (yellow
and red) interact, the DB and AD are
brought into proximity and can
activate transcription of reporter
gene.
Two-hybrid assay (interaction trap):
* používají se hostitelské kmeny kvasinek (např.
EGY48, EGY191) exprimující reportérový gen a
dále gen pro rezistenci, např. LEU2, který
umožňuje růst na médiu bez obsahu Leu; oba
geny jsou pod kontrolou operátoru LexA
* prvním krokem je příprava ,,bait" proteinu
fúzovaného s LexA a transformace kvasinek tímto
konstruktem;
* selekce kmenů, ve kterých bait protein
nepůsobí jako aktivátor, ani jako represor
transkripce reportérového genu;
* příprava cDNA knihovny, nebo specifických
proteinů fúzovaných s aktivační doménou (B42 pro
LEU2 a LacZ);
Point mutations in the LXCXE
motif disrupt the AhR-pRb
interaction. Site-directed
mutations were introduced
into the AhR to substitute
each of the conserved
residues in the LXCXE motif
with an alanine.
Yeast cells containing a LacZ
reporter plasmid (p8op-LacZ)
were transformed with the
wild-type (WT) or various
alanine-substituted AhR bait
constructs (L331A, C333A,
E335A) in the absence (+pRb)
and presence (-pRb) of plasmid
pB42ADpRb and grown on
leucine-deficient X-gal plates
at 30°C for 3 to 5 days.
[35S]AHR was synthesized in vitro
in the presence (T) or absence (0)
of 100 nM TCDD. Aliquots of the
unfractionated translation
products (35SAHR) or of two
successive effluents from control
nickel-agarose columns (Cont-1 and
Cont-2) or RB-bearing columns
(pRB-1 and pRB-2) were analyzed
by SDS-PAGE.
Protein-protein interactions between AHR and RB.
Aliquots of 1 mg each of whole cell
lysates were incubated with
monospecific rabbit anti-RB
antiserum, anti-AHR antiserum, or
control rabbit polyclonal serum.
Bound proteins were recovered with
protein A plus protein G-Sepharose,
washed, eluted by boiling, and
analyzed by SDS-PAGE and
immunoblotted with anti-RB and anti
AHR antibodies
SRC-1 mutants were fused to glutathione S-transferase and expressed
in Escherichia coli. Expressed fusion proteins from bacterial cell lysates
were put on glutathione-agarose beads. Protein coupled to beads was
incubated with in vitro-translated [35S]methionine-labeled ARNT or RIN1,
and absorbed proteins were subjected to SDS-PAGE. The two left lanes
contained 1 l of in vitro-translated RIN1 and 1 l of in vitro-translated
mARNT (25% input) for comparison.
FRET
FRET ­ příklad:
Analysis of ligandinduced
YFP-ER-CFPER
interactions in MCF7
cells.
A, representative FRET
images. Images
were acquired in COS-1
cells transfected with
YFP-ER and CFP-ER.
Interakce proteinu s DNA
* příprava cytosolového nebo jaderného extraktu;
* electrophoretic mobility shift assay ­ EMSA (gel
retardation assay);
* methylation interference; DNAse I protection
assay ,,footprinting"
EMSA
* příprava značené DNA próby (end labeling, nick
translation)
* binding reaction (jaderný nebo cytosolový extrakt,
carrier DNA a značené DNA próby) loading
buffer
* nedenaturující PAGE
* vysušení gelu a autoradiografie
EMSA
EMSA
Kontrola specifity
DNase I foot printing analysis
A DNA fragment (117 bp
for the coding strand and
112 bp for the non-coding
strand) containing a single
copy of HRE or XRE in the
central part and incubated
with DNase I. The digeste
DNA was precipitated with
ethanol and separated in
the denaturating
acrylamide gel containing
8 M urea.
Identifikace sekvencí, na které se vážou proteiny:
* např. XRE (DRE) - N T/G T GCGTG A/C G/T A/T A/G N
* genome-wide analysis
! Řada těchto sekvencí
neaktivuje transkripci !
Reporter gene assays
* stabilní
* transientní; kontrola ,,transfection efficiency"
(Renilla luciferase, GFP, -gal)
* důležité vlastnosti:
* hladina proteinu = hladina mRNA
* snadno detekovatelný produkt
* nesmí se nacházet v cílové buňce
* nesmí ovlivňovat cílovou buňku
Plasmid vectors
Reportérové geny:
GFP reporter
Luciferase reporter
* možnost sledovat dynamiku odpovědi
-Galactosidase reporter
- možnost využití k in vivo experimentům;
Expression domains of Hox genes in a mouse. The photographs show whole
embryos displaying the expression domains of two genes of the HoxB complex
(blue stain). These domains can be revealed by in situ hybridization or, as in
these examples, by constructing transgenic mice containing the control
sequence of a Hox gene coupled to a LacZ reporter gene, whose product is
detected histochemically. Each gene is expressed in a long expanse of tissue
with a sharply defined anterior limit. The earlier the position of the gene in its
chromosomal complex, the more anterior the anatomical limit of its expression.
Thus, with minor exceptions, the anatomical domains of the successive genes
form a nested set, ordered according to the ordering of the genes in the
chromosomal complex.
Thin layer chromatography
­ + +
CAT reporter
* nevýhodou je radioaktivní detekce; často
pracná
Proteiny (2):
* funkce jednotlivých domén;
* posttranslační modifikace (fosforylace,
acetylace, glykosylace);
* regulace odborávání proteinu (ubikvitinace;
proteazomální degradace);
* manipulace s proteinem (overexprese;
dominant negative constructs, antisense
oligonucleotides, siRNA).