Biochemistry and Molecular Biology

BIOC:3110 Biochemistry 3 s.h. One-semester survey of basic concepts in modern biochemistry and molecular biology; emphasis on application of biochemical concepts to human metabolism; appropriate for students who plan to pursue a career in health care or want an overview of biochemistry as a discipline. Requirements: one year each of college-level biology and chemistry. Recommendations: one semester of organic chemistry.

Several forward and reverse proteomic approaches are available that can be used to identify interaction partners for a protein of interest. Here we provide methods for identifying interacting partners by the yeast two-hybrid system (a reverse proteomic method) and by tandem immuno-affinity purification of protein complexes combined with mass spectrometry (a forward proteomic method).

The yeast two-hybrid system
The yeast two-hybrid system is a convenient reverse proteomic approach to identify and study protein-protein interactions (Fields and Song, 1989;Vidal and Legrain, 1999;Walhout and Vidal, 2001). The yeast two-hybrid system can be used with a single bait protein of interest. However, large datasets in which many C. elegans proteins were used as baits are now also available (Boulton et al., 2002;Davy et al., 2001;Li et al., 2004;Tewari et al., 2004;Walhout et al., 2002;. The yeast two-hybrid system is based on the functional reconstitution of an intact transcription factor that activates reporter gene expression ( Figure 1). Reporter gene expression can efficiently be selected for in yeast. As indicated by the name, the yeast two-hybrid system utilizes two hybrid proteins. The bait protein (X; Figure 1) is fused to the DNA binding domain (DB; Figure 1) of a transcription factor. The prey protein (Y; Figure 1) is fused to the transcription activation domain (AD; Figure 1) of a transcription factor. In our system, we use the yeast transcription factor Gal4. When two hybrid proteins are co-expressed in yeast and when X and Y can physically interact with each other, a functional transcription factor is reconstituted. This reconstituted transcription factor activates the expression of a set of reporter genes. To minimize the number of false positives identified, we use 3 reporter genes: HIS3, URA3 and lacZ. Expression of these genes is measured by growth on minimal media lacking histidine (for HIS3), or uracil (URA3), or by a colorimetric ("blue-white") assay. Performing yeast two-hybrid screens consists of the following steps: 1) creating a DB-X bait, 2) testing self-activation by the DB-X bait, 3) performing the yeast two-hybrid screen and 4) identifying the prey. These steps are described below in more detail. For additional information see Vidal and Legrain (1999) and Walhout and Vidal (2001).

Creating DB-X baits
Bait-encoding ORFs can be cloned into a DB-containing vector by regular, restriction-enzyme based cloning or by Gateway cloning. For regular cloning, we use the plasmid pPC97 that has the DNA binding domain of Gal4 immediately upstream of a multiple cloning site (Vidal and Legrain, 1999). We generally prefer to use Gateway cloning because many DB-ORF baits can be created simultaneously and in a standardized and high-throughput manner . After creating the DB-ORF clone(s) of interest, they need to be transformed into the yeast strain MaV103 (Vidal, 1997;Vidal and Legrain, 1999;Walhout and Vidal, 2001). A protocol for high-efficiency yeast transformations is provided below. Because DB-ORF-containing plasmids contain a LEU2 marker gene, transformants need to be selected on plates lacking leucine (Sc-Leu). Before proceeding with the actual yeast two-hybrid screen, bait strains need to be examined for self-activation, i.e. their ability to activate reporter gene expression in the absence of an AD-Y interaction partner (see below).
For Gateway LR cloning the following reagents are required: 1. An Entry clone from the ORFeome that contains the ORF(s) of interest (Reboul et al., 2003). These can be obtained from Dr. Marc Vidal's laboratory, see http://worfdb.dfci.harvard.edu/.
2. A Destination vector containing the DNA binding domain of Gal4: (pDEST ; . This DB vector can be obtained from Invitrogen, as part of their Gateway-compatible yeast two-hybrid system (catalog number: 10835-031).

Testing self-activation by DB-X baits
1. After transformation to create the DB-X bait strain, pick 12 individual yeast colonies and patch them onto a fresh Sc-Leu plate. We patch 5 control strains at the bottom of each plate for comparison (see Walhout and Vidal (2001) for details).
3. Next day, replica plate the patches onto 5 Sc-Leu,-His + 3-amintotriazole (3AT) plates (containing 20, 40, 60, 80 and 100 mM 3AT). 3AT is a competitive inhibitor of the His3 enzyme and only baits that exhibit self-activation will be able to grow in the presence of this compound. After replica plating, the 3AT-containing plates need to be replica cleaned 2 or 3 times, until no yeast is visible (Walhout and Vidal, 2001).
4. Next day, perform lacZ assay (see below for protocol).
5. Look at the 3AT plates on days 3, 4 and 5 after replica plating. Bait strains that grow on plates containing 100 mM 3AT cannot be used in yeast two-hybrid experiments. For baits that can be used, use the lowest 3AT concentration at which no growth is observed after 5 days in subsequent yeast two-hybrid screens.
6. We also perform lacZ assays to examine self-activation (see below). Patches that are blue correspond to self-activating baits.

Performing yeast two-hybrid screens
1. For yeast two-hybrid screens, the transformation protocol indicated below should be followed. For screens, we use 30 µg of a C. elegans AD-Y cDNA library . This library contains cDNAs obtained from worms of all developmental stages, males and dauer larvae and is highly comprehensive . The vector in which the cDNAs were cloned (pPC86, Vidal, 1997) contains a TRP1 marker for selection of yeast transformants on media lacking tryptophan. The transformation reactions should be directly plated on Sc-Leu,-His,-Trp + 3AT plates to select for colonies in which a protein-protein interaction between a bait and prey protein takes place.
2. To estimate the transformation efficiency of the reaction, serial dilutions of one reaction (1/10; 1/100 and 1/1000) should be plated on Sc-Leu,-Trp media (Walhout and Vidal, 2001). After incubating for 3 days at 30°C, the number of colonies should be counted and the total number of transformants calculated. We aim to screen at least 1 million colonies per bait.
3. Incubate the screen plates for 4-5 days at 30°C. 4. Pick any growing colonies (that are clearly bigger than background colonies, this varies from bait to bait) and patch with a sterile toothpick on a fresh Sc-Leu,-Trp,-His + 3AT plate. Incubate overnight at 30°C.
5. Next day, replica plate the positives onto a fresh Sc-Leu,-Trp,-His + 3AT plate, a YEPD plate containing a nitrocellulose filter for lacZ assays (see below) and a Sc-Leu,-Trp,-Ura plate. Replica clean the 3AT containing plate and the Sc-Leu, -Trp,-Ura plate until no yeast is visible. Incubate at 30°C.
6. Next day, perform the lacZ assay (see below).
7. Examine growth of potential positives on the other two plates after incubating for 3-5 days. Any growing patches are considered positives. We only consider colonies that score positive for at least 2 reporter genes, e.g., they grow on 3AT-containing media and are blue.
8. Retest each interaction in fresh bait-containing yeast cells by PCR/Gap-repair (Walhout and Vidal, 2001) to minimize the number of false positives.

Yeast transformations
1. Patch the required yeast strain on a YEPD plate and incubate overnight at 30°C.
2. Next day, inoculate 500 ml liquid YEPD media with the relevant yeast strain to a density of OD = ~0.1.   18. Resuspend the cells in 350 µl sterile water and plate on the appropriate selective media (i.e., Sc-Leu,-Trp,-His with the appropriate concentration of 3AT; Walhout and Vidal, 2001).

lacZ assays
1. Yeast should be grown overnight at 30°C on a nitrocellulose filter that is placed on top of a YEPD plate (Walhout and Vidal, 2001). We use filters from Osmonics (catalog number: WP4HY13700).
4. Take the nitrocellulose filter containing the yeast with a pair of tweezers and quick-freeze for 10 seconds in liquid nitrogen. Thaw while holding with the tweezers.
5. Carefully place the nitrocellulose on top of the Whatman filters, with the yeast patches facing up.
7. Blue yeast patches correspond to self-activating baits (when only DB-X is used), or to potential positives (when DB-X is screened against an AD-Y cDNA library).

Identifying interacting preys
The ORFs encoding interacting preys can be obtained from yeast by colony PCR using AD and Term primers. For a protocol see: Walhout and Vidal (2001). PCR products can be used in PCR/Gap-repair to retest potential interactions. PCR products corresponding to retesting positives should be purified to remove the primers. Subsequently, PCR products can be sequenced using the AD primer to obtain an IST (interaction sequence tag). ISTs can be blasted against Wormpep in Wormbase (http://www.wormbase.org/db/searches/blat) to identify the prey.

Tandem immuno-affinity purification
Multi-protein complexes are the functional units of many cellular processes. Defining the components of these complexes, how they associate and their intrinsic biochemical activities provides a wealth of information about the context in which proteins operate (Deshaies et al., 2002;Gavin et al., 2002;Ho et al., 2002;Rigaut et al., 1999;. Here we describe a standardized method for purification of protein complexes from C. elegans extracts by tandem immuno-affinity (Polanowska et al., 2004). This method describes the following steps: 1). Creating the transgene for expression of an epitope tagged form of your protein of interest (P.O.I.), 2). Growth and harvesting the transgenic line, 3). Generating lysates, 4) Preparing antibody coupled beads, 5) Tandem immuno-affinity purification of protein complexes, and 6) Preparation of the sample for mass-spectrometry (MS).

Creating the transgene
We have constructed pSB_GW::TAG, a Gateway destination vector that can be used to generate transgenic animals that express any P.O.I. fused at the C-terminus to HA_8xHis_TEV_Myc epitopes, enabling associated protein complexes to be purified from whole worm or embryo extracts by tandem immuno-affinity (Figure 2A; Polanowska et al., 2004). See  for details on Gateway cloning. The promoter and coding region of a gene of interest (G.O.I.) are cloned into the Gateway entry vector, sequenced, and then transferred by Gateway LR cloning into pSB_GW::TAG as previously described (Polanowska et al., 2004; Figure 2A). Stable transgenic animals generated by microparticle bombardment (Praitis et al., 2001) of unc-119(ed3) mutants with pSB_G.O.I::TAG are then tested for transgene expression by western blotting using antibodies against either 1) the HA epitope (MAb12CA5), 2) 8xHis (anti-His antibody, Amersham BioSciences), or 3) the Myc epitope (MAb9E10). O.I) into p221 using the BP reaction, and from p221 into pSB_GW::TAG using the LR reaction. An example of this is shown on the right in which universal primers have been used to PCR amplify the promotor and coding region (P+C) for Y41E3.9 from pSB_Y41E3.9::TAG (B) Microparticle bombardment is used to integrate the construct into unc-119(ed3) mutants at low copy to generate a transgenic line expressing the protein of interest (P.O.I.) fused to C-terminal epitope tags. The transgenic line is grown to high density in a BioFlo5000® fermenter, harvested and extracts generated for purification purposes(C). Tandem immuno-affinity chromatography over protein-A-MAb12CA5 and then protein-A-MAb9E10 is used to purify the P.O.I.-associated protein complex, before complex components are identified by mass-spectrometry. An example on the right shows a silver stained 4-12% gradient gel comparing the mock purification after (1.) HA and (2.) HA + Myc to the (3.) Y41E3.9 associated protein complex after HA + MYC immunoaffinity.

Growth and harvesting the transgenic line
We have optimized large-scale production of transgenic lines in BioFlo5000® fermenters (New Brunswick Scientific) or alternatively, smaller scale cultures grown in S-basal may yield sufficient material for purification purposes. See elsewhere for details (Polanowska et al., 2004). The weight of worms required for purifying a given protein complex to sufficient levels for protein identification by MS will depend on the level of expression from the genes promotor and must be determined for each transgene. The complexes we have studied so far function in the DNA damage response and are expressed at low levels in all proliferating cells, but are undetectable in somatic tissues. For all complexes (n=5) studied approximately 50g of biomass was required to obtained sufficient protein for MS.
To prepare the worms for lysis: