A key component of gene functional analysis and drug target discovery is an understanding of how proteins interact within the cell. The ProQuest™ Two-Hybrid System with Gateway® Technology is a tested, robust, in vivo yeast-based system for identifying protein-protein interactions. It provides specially engineered components for bait and prey expression, and enables the rapid movement of sequences between the ProQuest™ system and other expression and analysis tools using simple recombinant cloning.

The ProQuest™ System vectors include the following features:

pDEST™ 32 bait vector

  • ARS-CEN sequence for low-copy number maintenance in yeast
  • attR sites for Gateway® cloning
  • LEU2 for selection of recombinants on medium lacking leucine

pDEST™22 prey vector

  • For the expression of a known gene of interest to generate a prey fusion protein
  • ARS-CEN sequence for low-copy number maintenance in yeast
  • attR sites for Gateway® cloning
  • TRP1 for selection of recombinants on medium lacking tryptophan

pEXP-AD502 prey vector

  • For the construction of a cDNA or genomic library for the identification of prey proteins
  • ARS-CEN sequence for low-copy number maintenance in yeast
  • attR sites for Gateway® cloning
  • LEU2 for selection of recombinants on medium lacking leucine

pEXP32/Krev1

  • A control bait vector, expressing Krev-1 (a member of the Ras family of GTP binding proteins) fused to the GAL4 DNA binding domain

pEXP22/RalGDS-wt

  • A strong positive interaction control prey vector, expressing wild-type RalGDS fused to the GAL4 DNA activation domain

pEXP22/RalGDS-m1

  • A second control prey vector, serving as a weak positive interaction control

pEXP22/RalGDS-m2

  • A negative interaction control prey vector
References
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  2. Richards MC, Heron SE, Spendlove HE, Scheffer IE, Grinton B, Berkovic SF, Mulley JC, Davy A, Novel mutations in the KCNQ2 gene link epilepsy to a dysfunction of the KCNQ2-calmodulin interaction. J Med Genet 2004 41(3):e35-e35
  3. Ueki N, Hayman MJ, Signal-dependent N-CoR requirement for repression by the Ski oncoprotein. J Biol Chem 2003 278(27)24858-24864
  4. Missero C; Pirro M T; Simeone S; Pischetola M; Di Lauro R; The DNA glycosylase T:G mismatch-specific thymine DNA glycosylase represses thyroid transcription factor-1-activated transcription. J Biol Chem 2001 276(36)33569-75
  5. Lu Q, Hope LW, Brasch M, Reinhard C, Cohen SN, TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation. Proc Natl Acad Sci U S A 2003; 100(13):7626-31