The first step in a yeast two-hybrid screen is to construct a bait plasmid that expresses the protein of interest as a fusion to the DNA-BD. This plasmid is transformed into the appropriate yeast reporter strain and a series of control experiments are then performed to establish whether the bait is suitable for use in a library screen or interaction studies between two known proteins.
The construction of a bait plasmid expressing the protein of interest is described in Subheadings 3.1.1-3.1.4. This includes a description of (1) the DNA-BD fusion vector; (2) cloning procedures; (3) transformation of yeast; and (4) testing the DNA-BD fusion construct.
The pGBKT7 (Fig. 1) DNA binding domain fusion vector (Clontech) expresses bait proteins fused to the C-terminus of the GAL4 DNA-BD. The multiple cloning site (MCS) in pGBKT7 contains unique restriction sites in frame with the 3'-end of the GAL4 DNA-BD allowing for the generation of GAL4 DNA-BD/bait protein fusions. In this configuration the bait protein is also expressed as a fusion to a c-Myc epitope tag (see Fig. 1 and Note 3).
DNA manipulations were performed by standard recombinant DNA methods described by Sambrook and Russell (3) and are not described in detail here because of space limitations. The target gene encoding the bait protein was PCR-amplified using a high-fidelity thermostable DNA polymerase and oligonucleotide primers containing Ndel and BamHI restriction sites (see Note 4). The blunt-ended amplified DNA fragment was subcloned into the Srfl site of the pPCR-Script vector (Stratagene, La Jolla, CA) followed by DNA sequence analysis using gene-specific and vector-specific oligonucleotide primers to ensure that no PCR-generated mutations had occurred. The cloned gene was then cut out from pCR-Script using Ndel and BamHI and ligated into the same restriction sites of the pGBKT7 vector. The DNA was transformed into E. coli DH5a cells by standard methods (3), plated onto LB medium containing kanamycin (50 ^g/mL), and incubated overnight at 37°C. Single colonies were selected and grown overnight in LB liquid medium containing kanamycin (50 ^g/ mL). The plasmid DNA was then isolated (4) and analyzed for the presence of the insert using restriction enzyme digestion. Using the T7 primer and the 3' DNA-BD sequencing primer (Clontech) flanking the MCS site in pGBKT7, the plasmid DNA was subjected to DNA sequencing to ensure that the gene of interest was in-frame with the GAL4 DNA-BD fusion partner.
The next step is to test the activity of the bait protein in yeast reporter strains. The bait plasmid and the empty DNA-AD (prey) vector (see Fig. 2 and Subheading 3.1.4.) must be transformed into the appropriate yeast reporter strain. The authors use AH109 and Y187 as the mating strains (see Subheading 3.2.2.) and it is recommended that the same tests (see Subheading 3.1.4.) be per-
A c-Myc epitope tag
A c-Myc epitope tag
MATCHMAKER 51 DNA-BO Victor 1155 iRE.n Saving ^plim.r^_ 6AU DM-Bi^s Di.m.in
TCA TCG GAA GAG AGT AGT AAC AAA GGT CAA AGA CAG TTG ACT G TA TÇG CCG GAA TTT
3-3:7_T7 Sequencing Primer ^
' TÎPnmmi , c-MycEpitnpaTsg
GTA ATA CGA CTC ACT ATA GGG CGA GCC GCC ATC ATG GAG GAG CAG AAG CTG ATC TCA GAG GAG G AC CTG
CAT ATG GCC ATG GAG GCC GAA TTC CCG GGG ATC CGT CGA CCT GCA GCG GCC GCA TAACTAGCATAACCCC
«del «col S/Ï1 tcrt I Small fl'™H 1 Salt PU 1 STOP stop
* T7 Terminator
TTGG G G C CTCT A A A C 6 G ETC TTG AG G G GTTTTTT G CG CG CTTG CAG CCAAG CT A ATTC CG G G C G A ATTT CTT ATG ATTT
ATG ATTTTT ATT ATT A A AT A A G TT AT A A A A A A AAT A AGTGT ATA C AAATTTT A AAG TGACTCTTAGG TTTT A A AAC G A A A A
3' ON A-en Sequencing Primer
MATCHMAKER 31 DNA-BD Vattot Insert ScifiGrting Amplpmsr
Fig. 1. Schematic diagram of the DNA-binding domain bait vector pGBKT7. The multiple cloning site is shown. (Printed with permission. ©1999 Becton, Dickinson and Company.)
formed in both strains. To do this only a few yeast transformants are required and the protocol described below can be used.
1. Inoculate 10 mL of liquid YPDA medium with a yeast colony and grow overnight at 30°C at 250 rpm in a rotary shaker.
2. Harvest 1.5 mL of yeast cells for each transformation in a microcentrifuge tube by pulse centrifugation for 5 s (see Note 5). Decant the supernatant by inverting the tube and shake once, leaving the cells in 50 to 100 ^L of the remaining liquid.
3. Add 2 ^L of 10 mg/mL carrier DNA and resuspend the cells with a pipet tip.
4. Add in order 1 ^g of bait plasmid DNA, 0.5 mL of PLATE mixture, and 20 ^L 1 M DTT. Vortex briefly after each addition.
Before starting any yeast two-hybrid screen it is important to test the DNA-BD/bait fusion for (1) transcriptional autoactivation, (2) protein expression, and (3) toxicity to the yeast cells.
Autoactivation of His reporter gene expression may be a problem with some proteins fused to the DNA-BD, which may lead to the identification of false-positive clones. It is therefore important to determine the level of leaky His reporter gene activity by the DNA-BD/bait fusion protein itself and to test whether this background autoactivation can be suppressed. Suppression of background growth on SD medium lacking His can be performed using 3-AT, a competitive inhibitor of the yeast HIS3 protein. Conditions are sought where the AH109 reporter strain expressing only the DNA-BD/bait fusion protein does not grow on SD medium lacking His.
1. Using the yeast transformation protocol outlined in Subheading 3.1.3., cotransform AH109 yeast cells with the DNA-BD/bait plasmid and the empty AD prey plasmid pGADT7 (Fig. 2).
2. Select for transformants on SD/-Trp/-Leu plates (see Notes 1 and 2) and grow up a colony overnight in 10 mL of the same SD media.
3. Plate 100 ^L of cells onto SD/-Trp/-Leu/-His plates containing increasing concentrations of 3-AT (0, 5, 10, 20, 30 mM). In addition, plate cells on SD/-Trp/-Leu as a control for growth.
4. Incubate plates at 30°C.
5. After 4 to 6 d examine the growth of colonies on each plate. If the bait strain exhibits background growth on SD/-Trp/-Leu/-His medium then it will be necessary to eliminate/reduce the background by adding 3-AT to the selection medium for the screen. We have found that concentrations of 5 to 30 mM are sufficient to select against autoactivation (see Note 7).
The expression of the fusion protein should be verified by Western blotting (see Note 8). pGBKT7 encodes a c-Myc epitope tag (Fig. 1) that can be used for immunological detection of the expressed fusion protein. Alternatively, commercially available antibodies raised to the DNA-BD may be used.
Total yeast protein extracts are prepared as described below:
1. Grow Y187 (bait) overnight to OD600 = 0.7 and harvest 1.5 mL of cells in a microcentrifuge tube.
2. Pulse-spin for 5 s in a microcentrifuge, discard supernatant, and wash once with water.
3. Pulse-spin for 5 s in a microcentrifuge and resuspend cells in 100 ^L of protein extraction sample buffer.
5. Centrifuge at 13,000g for 5 min.
6. Load 15 ^L per lane on a standard SDS polyacrylamide gel. Methods for pouring and running SDS gels as well as Western blotting can be found in ref. 3.
Compare the growth rate in liquid culture of yeast containing the bait-DNA-BD fusion and yeast containing the empty DNA-BD fusion vector. If growth is noticeably slower then the bait, fusion protein may be toxic to the yeast cells (see Note 9).
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