The "Two-hybrid System" for detecting protein

The "Two-hybrid System" for detecting protein-protein interactions in yeast
An easy and powerful system for detecting noncovalent protein-protein interactions in
vivo in yeast was developed independently by Stan Fields and Roger Brent about 10
years ago. Since that time many researchers have discovered and reported new
connections between proteins using their techniques. Since many biological processes are
carried out in protein complexes formed by noncovalent bonds, knowledge about proteinprotein interactions is extremely useful to understand how these complexes are structured
and how they work.
The principle of the experiment is based on the modular nature of transcription
factors. Certain transcription activating proteins act by (1) binding to a special DNA
sequence near the promoter, and (2) doing something that is still mysterious to activate
transcription from the promoter. The part of the transcriptional activating protein that
binds DNA (the DNA binding domain or DBD) can be separated from the rest of the
protein and will still bind to DNA, but will not activate transcription. The activating part
of the protein can also be separated and attached to a different DBD, and will activate
transcription of a different set of promoters, but will not activate transcription if it is not
attached to a DBD. The Fields system relies on the GAL4 transcriptional activator (which
contains both a DBD and an activation domain), whereas the Brent system relies on nonyeast proteins like E. coli lexA (for its DBD) and Herpesvirus VP16 (for its activation
The expression of a DBD covalently fused to an activation domain (as it is in the normal
GAL4 protein) leads to expression of genes to which the DBD can bind (In wild type
yeast, the GAL genes). The normal GAL genes are not convenient to assay in terms of
their expression so we use “reporter genes” whose activity is easy to see. The expression
of these "reporter genes" report that the transcription factor is functioning. If a separate
DBD and a separate activation domain are expressed in the same cell and they do not
associate, no functional activator protein is made and no reporter gene expression is
detected. This allows for testing for noncovalent interactions as follows. If the DBD is
joined to a protein of interest X and the activation domain is joined to a protein Y thought
to bind noncovalently to X, and these two, hybrid proteins (get it?, two-hybrid) are both
expressed in the same cell, they can bind and form a protein complex that both binds
DNA and activates transcription (even though X and Y may have nothing to do with
transcription, here they only serve to bring together the DBD and the activation domain).
Looking for signs of reporter expression will tell whether X and Y associate.
Notice that an absence of reporter function does not mean that X and Y do not associate.
A negative result cannot be interpreted (As Carl Sagan was fond of saying about aliens,
"Absence of evidence is not evidence of absence."). The fusion protein could be unstable,
or not correctly folded or have no DNA binding activity or activation activity for other
reasons, or might not be transported to the nucleus, etc., even though in real life, X and Y
might be associated tightly. Only a positive signal can be taken as a sign of association.
For the same reasons it is difficult to use the amount of reporter expression as an
indication of the strength of the interaction.
The proteins of interest X and Y can come from any source; they need not be yeast
proteins. In fact many studies using the two-hybrid system test mammalian cell proteins.
It is also possible to search for new proteins that interact with a protein of interest X by
screening libraries of sequences fused to the activation domain. Reporter expression
reveals members of the library that interact with X and these can be recovered and
Today we will begin a demonstration experiment using the two-hybrid system of Fields
to test for interactions between and among numerous different yeast splicing proteins that
are involved in recognition of the pre-mRNA branchpoint region and the addition of the
U2 snRNP to the assembling spliceosome.
Outline of the Experiment
The class will be provided with plasmids encoding two-hybrid proteins made by fusing
yeast splicing factor genes to the DBD and/or the activation domain of GAL4. There are
18 plasmids, 9 of them (the pAS plasmids) will be transformed into yeast strain Y187
using the TRP selectable marker on pAS, and the other 9 (the pACT plasmids) will go
into Y190 using the LEU selectable marker on the pACT plasmids. Transformants of
each Y187 strain will be mated to transformants from every Y190 strain on a 9 by 9 grid,
selecting for diploids that carry both plasmids on YMD-leu, -trp. After mating the
diploids will be replica plated to YMD-his +25mM 3-AT (3-amino-1, 2, 4, triazole is an
inhibitor of the HIS3 enzyme and is necessary because the reporter is "leaky") for testing
expression of a HIS reporter, and to YMD-leu,-trp for a beta-galactosidase assay to test
expression from a lacz reporter.
3-amino-1,2,4-triazole (3-AT) competitively inhibits imidazole glycerol phosphate dehydratase, a His
biosynthetic enzyme (Hilton et al., 1965; Klopotowski and Wiater, 1965; Wiater et al., 1971), and therefore
can limit histidine biosynthesis and growth. For twohybrid screens that use a HIS3 reporter, 3-AT is
employed – the HIS3 gene encodes the enzyme activity inhibited by 3AT. High expression of this reporter
gene, which arises from a successful two-hybrid interaction, can overcome the growth-inhibitory effect of
3-AT in the medium. When 3-AT is added to yeast media (1-10 mM) it will limit histidine biosynthesis and
is used in two-hybrid screens to "fine tune" leaky expression of the HIS3 reporter gene.Therefore, the use
of 3-AT and the HIS3 reporter enables positive selection for successful two-hybrid interactions.
Reporter expression will show which splicing proteins interact with which others. The
proteins we are testing are BBP, CUS1, CUS2, HSH49, HSH155, MUD2, PRP9, PRP11,
PRP21, SUB2, and as a control, URA3.
On Day 1 we will transform Y187 with the pAS plasmids to Trp independences, and
Y190 with the pACT plasmids to leucine independence. Everyone should do at least one
and the plates should be incubated at 30°C. I may ask some of you to do more than one.
You will use transformation method 2, except that the competent cells will have already
been prepared
The following lab day, assuming all the transformations went as planned, each team will
streak two YPAD plates, one plate with 5 of the Y187 transformants (one of each of 5
plasmids) and one plate with each of 5 of the Y190 transformants. After these grow
overnight, they are stamped on the same velveteen at right angles, and then to a YPAD
plate for mating as we did before. These two strains did not mate that well last year, so
we should let them grow together at least overnight. Then the YPAD plate should be
replicated to an SCD-trp-leu plate to select for Y187/Y190 diploids. We will need to
make sure we have all strains containing all combinations of the pairs of plasmids. This
SCD-leu-trp mating selection plate will be the master plate to make replicas for the beta
gal assay, and to an SCDhis+3AT plate to test for HIS+ growth due to activation of the
HIS reporter. We will check growth on SCD-his+3AT, and after the replicated SCD-trpleu plate grows, we will overlay it with agarose containing X-gal, to assay for betagalactosidase enzyme, the product of the lacZ reporter.
Genotypes of Y187 and Y190
Y187 MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4Δ, met-, gal80Δ,
Y190 MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4Δ, met-,
YM Plates/media
YM Media
Yeast Nitrogen Base
6.7 g
(without amino acids)
20 g
(Wash before using)
900 ml
Separately sterilize
20 g
100 ml
Autoclave solutions 1 and 2 separately
Mix the two thoroughly and then pour plates (25 ml per plate)
0.5M LiAc
50 mM Tris HCl pH 7.5
Final volume
Filter sterilize and store RT
50ml of 1M stock
5ml of 1M stock
1ml of 0.5M stock
Dilute the 5x TEL with sterile water to 1x and use
50% PEG 4000
Dissolve 50g PEG4000 in 100 ml water.
Filter sterilize and store
80ml 50% PEG4000
20 ml 5xTEL
Salmon sperm DNA
Dissolve in water to 10 mg/ml
Sonicate 1 min. x3
boil 10 min. and aliquot.
Grow strain in 10 ml YPD overnight
Next day measure OD 600
Inoculate 100ml YPD with overnight culture so that OD is 0.1/ml (i.e. 10 OD of cells)
Grow till OD = 0.8 30C
Spin down cell 3 min 3000 rpm
Resuspend 10 ml 1xTEL and shake overnight at Room temp
Next day spin down cells 3 min 3000 rpm
Resuspend in 1 ml 1xTEL.
Leave 30 min. on the bench at Room temp.
In a eppendorf tube add
Sonicated Salmon Sperm DNA (10 mg/ml)
5 ul
DNA (0.2 ug)
5 ul
Competent Cells
100 ul
Mix by pipeting up and down
Incubate room temp without shaking for 30 min.
Add 700 ul 40% PEG/TEL
Mix by pipeting up and down
Incubate room temp without shaking for 60 min.
Heat shock 42C 10 min.
Spin cells gently (setting 7 in Eppendorf microfuge) for 10 sec.
Resuspend cells in 1 ml sterile water and plate on plates selecting for the plasmid. You
want to get about 200 colonies per plate.
Grow 30C for two days.
LacZ overlay assay
Grow your yeast on selective plates until colonies are 1.5-2 mm or so.
Melt a solution of 1% agarose by microwaving at low power (TAKE OFF THE CAP!).
Cool to 55°C in the water bath.
Prewarm some 1 M Na PO4 pH7.0 to 55°C.
Prewarm a 50 ml plastic test tube.
You will need 5 ml of 1% agarose, 5 ml of Na PO4 pH7.0, 100 ul 10% SDS, and 200 ul
of 10% X-gal in dimethyl formamide (DMF) per 90 mm petri dish. (NB less X-gal will
work for strong expressing clones, but KEEP the DMF amount constant!)
SDS and DMF are essential for permiablizing the yeast cells.
Mix components in the following way to avoid precipitation:
Put 5 ml molten agarose into the prewarmed test tube. Add 100 ul SDS, swirl to mix.
Put 5 ml prewarmed Na PO4 pH7.0 into the agarose-SDS and mix.
Add 200 ul X-gal in DMF, mix.
With a disposable pipette, draw as much of the molten mixture up as possible and
carefully apply it to the surface of the petri plate. Adding it too fast may disturb the yeast
on the plate, too slowly and the mixture may solidify.
After the overlaid mixture solidifies (a couple of minutes), put the plate at 37°C.
Strong lacZ expressing strains will turn blue within an hour. Plates can be incubated
longer to detect weaker lacZ expression.
It is possible to use less X-gal, but keep the DMF constant (e. g. can use 200 ul of a 5%
solution of X-gal in DMF).