Methods and reagents: False positives from the yeast two-hybrid system Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses false positives found when using the yeast two-hybrid system to isolate interactive proteins. For details on how to partake in the newsgroup, see the accompanying box. Characterization of protein-protein interactions is often facilitated by use of a two-hybrid protein system developed in yeast. This system is based on the interaction of proteins synthesized from two hybrid genes that are constructed in vitro and then transferred into and maintained within a cell on two separate, but compatible plasmids. One of the hybrid genes is made by fusing the DNA coding region of a well-characterized DNA-binding protein to a segment of DNA encoding a `bait' protein fragment. The other hybrid is made by creating a library of cloned DNA fragments within a vector that allows translational fusion of inserts with a known transcriptional activator. When the plasmids are co-resident in a cell that has one or more reporter genes with upstream binding sites specific for the DNA-binding protein, any interactions of the unknown fusion product with the bait protein will bring the transcriptional activator close to the promoter of the reporter gene. With the correct positioning of the activator, there will be an alteration in the level of reporter gene activity. Increased expression of the reporter is taken as an indication that the unknown gene fusion product, also known as the `prey', is able to interact with the bait protein, therefore placing the activator in contact with those factors necessary to stimulate transcription. In most cases, the well-characterized yeast transcription factor GAL4 and the DNA-binding domain of Escherichia coli LexA are used in constructing the fusions, and the E. coli LacZ gene is often used as the reporter [1,2]. More information can be obtained from http://xanadu.mgh.harvard.edu/ Praying for Prey **************** One netter wrote that after having spent a considerable amount of time characterizing a cloned gene obtained through screening a genomic library with a yeast two-hybrid system, the protein he found interacting with his bait turned out to be a false lead. Netters have had complaints about false positives in the yeast two-hybrid system for some time, and one person quite familiar with the problem, Erica Golemis (EA_Golemis@fccc.edu), wrote that this kind of trouble is not uncommon. Her lab has devoted an enormous amount of time and energy in collecting numerous accounts of false positive clones obtained through the use of a yeast two-hybrid system called the `Interaction Trap system'. A survey of 223 investigators conducted by Ilya Serebriiskii (ilya@scfuzzy.rm.fccc.edu) between October 1994 and January 1996 revealed that the problem of false positives is widespread. Their team has entered the relevant results from 100 library screenings into a database now available on the Web. From these, only 54 searches revealed biologically relevant interactors according to the investigator's estimations, with only four researchers reporting that no false positives were found, and 13 reporting that no preys worthy of further investigation were found. Interestingly, of the 73 false positives sequenced, the most common ones were found to be heat shock proteins (16), ribosomal proteins (14), cytochrome oxidase (5), other mitochondrial proteins (3), proteasome subunits (4), ferritin (4), transfer-RNA synthase (3), collagen-related proteins (3), zinc finger-containing proteins (3), vimentin (2), inorganic pyrophosphatase (2), and most surprisingly, proliferating cell nuclear antigen (PCNA) (2). The rest of those reported were not isolated more than once. The descriptions of the baits used and the false positives found are available from http://www.fccc.edu/research/labs/golemis/Table1.html. As quarterly updates to the false positive database are planned, more information should become available as more workers post their findings. The news for yeast lovers is somewhat encouraging because the protein being searched for by that unlucky netter was eventually identified by using a yeast two-hybrid system and a different bait. Unfortunately for him, however, someone else beat him to the punch. The hard lesson learned is that perhaps one should use multiple baiting attempts when designing experiments using this system, choose the bait very carefully and do plenty of controls. Given the 50% success rate reported in the survey, a minimum of two different baits should be used if one can afford it. GST fusion update ***************** In response to the Methods and reagents article concerning contaminants found when purifying GST fusion proteins (TiBS 21, 400-401), one netter wrote that one contaminant often co-purifying with GST fusions is the 60 kDa E. coli GroEL heat-shock protein (chaperonin 60). The contaminating protein was eliminated from the preparation by washing the affinity column with 5 mM ATP, or with a combination of GroES and ATP [3,4]. Pharmacia have also indicated that a 70 kDa protein co-purifying with many GST fusion products might be from the E. coli gene dnaK [5]. They suggest that the association of this degradative protein with fusion products can be disrupted by incubating them in 50 mM Tris (pH 7.4), 10 mM MgSO4, 2 mM ATP for 10 mins at 37 degrees C. Someone else asked about how to resolve dimers formed between GST fusion proteins. Although this was not mentioned in the previous article, the paper cited [6] shows that dimers formed between CD2 domains were stable even after being cleaved with thrombin to remove the GST fragments. However, when the CD2 fragments were denatured with 3M guanidine HCl and allowed to renature, the cleaved CD2 proteins behaved normally as monomers and were able to be resolved. Furthermore, it was thought that the CD2 proteins could form dimers by misfolding and that the misfolding was caused by the dimerization of the attached GST fragments. Bring me my BLOTTO ****************** Netters are always looking for a cheap alternative supplies. One discussion recently surfaced about the use of blocking agents for reducing background in western blots. Heat-treated bovine serum albumin (BSA) or purified casein has traditionally been used for this, however, they are becoming increasingly expensive. Several netters wrote that the BLOTTO (Bovine Lacto Transfer Technique Optimizer) technique works very well [7]. When blocking, netters say that a solution containing 5% w/v non-fat (less than 1% w/w fat) dry milk purchased from a local grocery store is an excellent alternative to BSA. Be careful though if you plan to use a biotin-streptavidin-based conjugation system, because some milk products contain biotin. Someone else mentioned that an alternative to BLOTTO is 5% v/v O'Darby's or Bailey's Irish Cream (BIC) liqueur [8]. BIC has the added advantage that the left-over mix can be used for celebration or commiseration, depending upon how the experiments turn out. Although BLOTTO has been shown to reduce undesirable contaminants that interfere with the PCR [9], BIC has not yet been tested for this. Boo Boo's on your blots *********************** A quick and inexpensive way to orient your gel blots when exposing them to X-ray film is to place fluorescent stickers on them. Recently, one netter suggested that glow-in-the-dark printed plastic bandage strips like those sold for children can be attached over one corner of your blotted membrane before exposing them. References ********** [1] Fields, S. and Sternglanz, R. (1994) Trends Genet. 10,286-292 [2] Gyuris, J. el al. (1993) Cell 75,791-803 [3] Thain, A. et al. (1996) Trends Genet. 12,209-210 [4] Sherman, M and Goldberg, A. L. (1994) J. Biol. Chem. 269,31479-31483 [5] Sherman, M. Y. and Goldberg, A. L. (1992) EMBO J. 11,71-77 [6] Murray, A. J. et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92,7337-7341 [7] Johnson, D. A. et al. (1984) Gene Anal. Tech. 1,3-8 [8] Elbrecht, A., Rowley, D. R. and O'Malley, B. W. (1987) BMBiochemica 4(2),12-13 [9] De Boer, S. H. et al. (1995) Nucleic Acids Res. 23,2567-2568 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/jan97.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1997. Methods and reagents: False positives from the yeast two-hybrid system. Trends in Biochemical Sciences 22(1):33-34. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************