High‐Throughput Screening for Protein‐Protein Interactions Using Yeast Two‐Hybrid Arrays

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Arrays are used for parallel assays of large numbers of proteins in a single experiment. They provide an alternative to libraries for protein screening purposes. This unit describes a protein array composed of living yeast (Saccharomyces cerevisiae ) cells that can be used in a functional screen for protein interactions, the two?hybrid assay. The protocols described here use the yeast proteome as an example, but they can be applied to any other genome or subset thereof.

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Basic Protocol 1: Construction of a Proteome‐Scale Protein Array for Yeast
Basic Protocol 2: Manual Screening for Protein Interactions Using a Yeast Protein Array
Alternate Protocol 1: Robotic Screening for Protein Interactions Using a Yeast Protein Array
Reagents and Solutions
Commentary
Figures

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Materials

Basic Protocol 1: Construction of a Proteome‐Scale Protein Array for Yeast

Materials

DNAs encoding proteins of interest (e.g., genomic DNA, cDNA library)
Appropriate primers for amplification of the protein‐coding sequences
Two‐hybrid plasmids: pOAD and pOBD2 (Hudson et al., ; Cagney et al., ; also see )
Nco I and Pvu II or other appropriate restriction enzymes, with buffers
recipeLiquid YEPD medium (see recipe )
S. cerevisiae host strains:
PJ69‐4A (MATa trp1‐901 leu2‐3,112 ura3‐52 his3‐200 gal4Δ gal80Δ LYS2::GAL1‐HIS3 GAL2‐ADE2 met2::GAL7‐lacZ ; James et al., )
PJ69‐4α (MATα trp1‐901 leu2‐3,112 ura3‐52 his3‐200 gal4Δ gal80Δ LYS2::GAL1‐HIS3 GAL2‐ADE2 met2::GAL7‐lacZ; Uetz et al., )
0.1 M lithium acetate
7.75 mg/ml salmon sperm DNA (Sigma), autoclaved (15 min at 121°C) and stored at −20°C
recipe96PEG solution (see recipe )
Dimethyl sulfoxide (DMSO)
35‐mm plates containing solid −Leu (for pOAD) and −Trp (for pOBD2) recipedropout medium (see recipe )
Liquid −Leu recipedropout medium (see recipe )
Single‐well microtiter plates (e.g., OmniTray, Nalge Nunc International) containing solid −Leu recipedropout medium (see recipe )
40% (v/v) glycerol
250‐ml conical flasks
30°C shaking incubator
8‐channel pipettor (to deliver 200 µl) and sterile pipetting troughs
96‐ and 384‐well microtiter plates (e.g., Nalge Nunc International)
Plastic adhesive tape for sealing plates
42°C incubator
Centrifuge with insert for 96‐well plates
Toothpicks, sterile
384‐Pin Replicator (Nalge Nunc International)
2‐ml cryotubes (e.g., Nalge Nunc International)

Additional reagents and equipment for PCR amplification ( appendix 4J ), agarose gel electrophoresis ( appendix 4F ), digestion of DNA with restriction endonucleases ( appendix 4I ), and gel purification ( appendix 4F ) and quantification ( appendix 4K ) of DNA

Basic Protocol 2: Manual Screening for Protein Interactions Using a Yeast Protein Array

Materials

20% (v/v) bleach (∼1% sodium hypochlorite)
95% (v/v) ethanol
Yeast protein array (see protocol 1 )
Single‐well microtiter plates (e.g., OmniTray; Nalge Nunc International) containing solid recipeYEPD +Ade medium (see recipe ), recipeYEPD medium (see recipe ), −Leu −Trp recipedropout medium (see recipe ), and −His −Leu −Trp +3AT recipedropout medium (see recipe )
recipeLiquid YEPD medium (see recipe )
DBD fusion–expressing yeast strain (see protocol 1 )
384‐Pin Replicator (Nalge Nunc International)
30°C incubator
250‐ml conical flask

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Figures

Figure 19.6.1 Two subsequent two‐hybrid screens of a full‐length activation domain–open reading frame library with full‐length PHO85 protein (a cyclin‐dependent kinase) as bait. (A ) First screen. (B ) Second screen. Reproducible positives are indicated by arrows. Some reproducible positives are either nonyeast contaminants (c) or false positives (f). Nonyeast contaminants can be identified by their unusual shape, size, or color. False positives are either nonreproducible or reproducible positives, but the latter are usually found in many independent screens with unrelated baits (hence they are nonspecific). Given these criteria, the following preys were identified as reproducible positives (plates are numbered in columns): PCL10, PCL6, CLG1 (plate 4), SOR1 (plate 5), CDC36 (plate 11), PCL9, PCL2, YPL229W (plate 12), YDL246C (plate 13), PCL8 (plate 14). PCLs are cyclins, and most of them are known PHO85 interactors; CLG1 is a cyclin‐like protein; CDC36 is a transcription factor; YPL229W is a protein of unknown function; SOR1 is sorbitol dehydrogenase; and YDL246C is a SOR1‐related protein. Most of these preys are either known or highly plausible interactors of PHO85. The biological role of an interaction between SOR1 and PHO85 remains unclear but is supported by YDL246C, which is a relative of SOR1. Note that these two screens have an unusually high number of contaminants on plates 9 and 10. The false positives have been found in more than 10% of all screens.

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Literature Cited

Literature Cited
	Cagney, G., Uetz, P., and Fields, S. 2000. High‐throughput screening for protein‐protein interactions using two‐hybrid assay. Methods Enzymol. 328:3‐14.
	Fields, S. and Song, O. 1989. A novel genetic system to detect protein‐protein interactions. Nature 340:245‐246.
	Flajolet, M., Rotondo, G., Daviet, L., Bergametti, F., Inchauspe, G., Tiollais, P., Transy, C., and Legrain, P. 2000. A genomic approach of the hepatitis C virus generates a protein interaction map. Gene 242:369‐379.
	Fromont‐Racine, M., Rain, J.C., and Legrain, P. 1997. Toward a functional analysis of the yeast genome through exhaustive two‐hybrid screens. Nature Genet. 16:277‐282.
	Hudson, J.R., Dawson, E.P., Rushing, K.L., Jackson, C.H., Lockshon, D., Conover, D., Lanciault, C., Harris, J.R., Simmons, S.J., Rothstein, R., and Fields, S. 1997. The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form. Genome Res. 7:1169‐1173.
	James, P., Halladay, J., and Craig, E.A. 1996. Genomic libraries and a host strain designed for highly efficient two‐hybrid selection in yeast. Genetics. 144:1425‐1436.
	Kraemer, B., Zhang, B., SenGupta, D., Fields, S., and Wickens, M. 2000. Using the yeast three‐hybrid system to detect and analyze RNA‐protein interactions. Methods Enzymol. 328:297‐321.
	Orr‐Weaver, T.L., Szostak, J.W., and Rothstein, R.J. 1981. Yeast transformation: A model system for the study of recombination. Proc. Natl. Acad. Sci. U.S.A. 78:6354‐6358.
	Rain, J.C., Selig, L., De Reuse, H., Battaglia, V., Reverdy, C., Simon, S., Lenzen, G., Petel, F., Wojcik, J., Schachter, V., Chemama, Y., Labigne, A., and Legrain, P. 2001. The protein‐protein interaction map of Helicobacter pylori. Nature 409:211‐215.
	Uetz, P., Hughes, R.E., and Fields, S. 1998. The two‐hybrid system: Finding likely partners for lonely proteins. Focus (Life Technologies) 20:62‐65.
	Uetz, P., Giot, L., Cagney, G., Mansfield, T., Judson, R., Knight, J., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., Qureshi‐Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rothberg, J. 2000. A comprehensive analysis of protein‐protein interactions in Saccharomyces cerevisiae. Nature 403:623‐627.
	Vidal, M. and Legrain, P. 1999. Yeast forward and reverse “n”‐hybrid systems. Nucl. Acids Res. 27:919‐929.
Key Reference
	Uetz et al., 2000. See above.
	This paper, the first to describe two‐hybrid arrays, provides the results of 192 screens and compares them to screens that used a library of preys.
Internet Resources
	http://depts.washington.edu/sfields/
	The Fields laboratory Web site contains more details on primer design, vectors, and array screening as well as results from several hundred screens.
	http://www.lab‐robotics.org/
	The Laboratory Robotics Interest Group Web site provides a guide to robotics manufacturers and product information.
	http://www.proteome.com
	Proteome's Web site includes a Yeast Protein Database.
	http://mips.gsf.de/proj/yeast/
	The Munich Information Center for Protein Sequences Web site provides yeast protein databases and information on other genomes and proteomes.
	http://www.fccc.edu/research/labs/golemis/InteractionTrapInWork.html
	The Golemis laboratory Web site offers general information on two‐hybrid screens and false positives.

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High‐Throughput Screening for Protein‐Protein Interactions Using Yeast Two‐Hybrid Arrays

Abstract

Table of Contents

Materials

Basic Protocol 1: Construction of a Proteome‐Scale Protein Array for Yeast

Basic Protocol 2: Manual Screening for Protein Interactions Using a Yeast Protein Array

Figures

Videos

Literature Cited