les_6 - 96 VI.4. GENETIC APPROACHES Genetic approaches rely...

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Unformatted text preview: 96 VI.4. GENETIC APPROACHES Genetic approaches rely on cDNA libraries that can be screened to find interacting proteins for your protein of interest. The main advantage of these screenings for PPI is the immediate availability of the cDNA encoding the identified binding partners, whereas in biochemically based approaches the genes encoding the interacting proteins still need to be cloned (except for some protein arrays such as NAPPA, DAPA,…) . In these genetic approaches, a bait is the protein that is used to screen your cDNA library with, in the search for preys to interact with the bait. VI.4.1. cDNA libraries There exist several kinds of cDNA libraries on the market. So before screening the protein of interest against a library, consider which type of cDNA library to use: 1. Choose a library in which your bait protein is expressed and functional. 2. There exist 2 main types of libraries (see also Table 6): - Classical cDNA libraries : - cDNAs prepared from the mRNA of one specific or diverse tissues, cells,… - based on oligodT or random primed cDNAs. With an oligodT cDNA library, you screen mostly against the C-terminus of proteins and may miss a lot of interacting partners (insert size!). This is not the case for random primed cDNAs, but they have the disadvantage that many hybrid proteins are expressed in the wrong reading frame or from the 5’ or 3’ untranslated regions of the mRNA, leading to non-natural proteins. --cDNAs from highly expressed mRNA’s are overrepresented, whereas cDNAs from lowly expressed mRNAs are rare - ORFeome libraries : 97 -=collection of individually cloned cDNAs comprising the full-length open reading frame ( ⇔ classical cDNA libraries), so absence of non-natural or incomplete proteins, no difference between low or highly expressed proteins -Mostly presented in an array form -Identity of the arrayed proteins is known, so no need for isolation and sequencing of the prey -pair-wise tests are possible Table 6. Preys in cDNA pools versus arrays of preys (ORFeome) In the following sections, we will discuss phage display, yeast two hybrid and variations . For FRET analysis we refer to chapter II on reporter systems. 98 VI.4.2. PHAGE DISPLAY VI.4.2.1.Principle In phage display technology, a whole foreign cDNA library is fused into the M13 bacteriophage genome. The resulting recombinant polypeptides are expressed and displayed on the surface of phage particles. Interacting proteins are identified as phage to bind to selected immobilized molecules. After washing, interacting phages are amplified and the DNA sequence of the putative interacting molecule is sequenced. The filamentous M13 bacteriophage is 1 μ in length. The single stranded circular DNA is stretched along the entire particle length and encodes for 11 proteins (g1p till g11p) and is coated with g8p protein (> 1000 copies). When extra DNA is inserted into the genome, the phage adapts by adding more p8 during assembly to accommodate the additional DNA. DNA....
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This note was uploaded on 05/28/2010 for the course WE BIBI000000 taught by Professor Johangrooten during the Spring '10 term at Ghent University.

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les_6 - 96 VI.4. GENETIC APPROACHES Genetic approaches rely...

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