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Yeast two hybrid assay ?


Yeast two hybrid assay: -
                                         Study interaction, detecting protein-protein interaction.

  Principle of the two-hybrid: -
A] Standard model of transcription activator: -
    - The DNA binding domain of an activator binds to an enhancer.
    - The activating domain interacted with the based complex. Recruiting it the promoter
     

B] Two hybrid assay for protein-protein interaction using gene clone technique linked the gene one protein to the part of a gene encoding a DNA binding domain, to encode 1 hybrid protein, linked the gene for another protein to the part of the gene encoding a transcription activating domain to encode a two hybrid proteins are introduce into yeast cells bearing the appropriate promoter enhancer and reporter gene (laczin) the two hybrid protein can get together and serve as an indicator activated transcription produces a bond and reporter gene product which can be detected with colorimetric assay using X-gal. (Blue colour compound)

For Example: -
 One hybrid protein contributes DNA bonding domain, and other contributes or transcription activity domain the two parts of activator are held together by the interaction between protein X and Y. if X and Y
Interact and X gal is use in the assay reporter protein gene product yeast cell will turn blue if X and Y do not interact no activator gene will occur in this case the yeast cell will remain while in the presence of X gal the gal four DNA binding domain and transcription activity possibility exist.
C] Two hybrid screen for a protein that interact with protein zinc yeast cell are transform in 2 plasmid encoding a DNA binding domain coupled to a bait protein the other is  a cell of plasmid containing many cDNA coupled to the coding region for a transcription activating domain each of this encode a fusion protein containing the activator domain fuse to and unknown DNA product each yeast cell is transform with just one of this pray encoding plasmid but several of their product are show together here for convince.
Once pray protein interact with the bait protein Z this brings together the DNA binding protein and the transcription activity domain so they can activate reporter gene.


                                                                          the yeast two-hybrid system for detecting protein-protein interaction. the target protein fused to DNA-binding domain (Bait) when this  target protein bind another specialy designed protein the cell nucleus (prey) theire interaction brings together and get transcription activate and the switches on the transcriptional reporter gene.

Reference: -



  •  Young KH (February 1998). "Yeast two-hybrid: so many interactions, (in) so little time". Biology of Reproduction. 58 (2): 302–11. doi:10.1095/biolreprod58.2.302. PMID 9475380.

    1. ^ Jump up to:a b c d e f g h i j k l m n o p q r s t u v w x Joung JK, Ramm EI, Pabo CO (June 2000). "A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions". Proceedings of the National Academy of Sciences of the United States of America. 97 (13): 7382–7. Bibcode:2000PNAS...97.7382J. doi:10.1073/pnas.110149297. PMC 16554. PMID 10852947.
    2. ^ Jump up to:a b c d e f g h i Hurt JA, Thibodeau SA, Hirsh AS, Pabo CO, Joung JK (October 2003). "Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection". Proceedings of the National Academy of Sciences of the United States of America. 100 (21): 12271–6. Bibcode:2003PNAS..10012271H. doi:10.1073/pnas.2135381100. PMC 218748. PMID 14527993.
    3. ^ Fields S, Song O (July 1989). "A novel genetic system to detect protein-protein interactions". Nature. 340 (6230): 245–6. Bibcode:1989Natur.340..245F. doi:10.1038/340245a0. PMID 2547163. Abstract is free; full-text article is not.
    4. ^ Jump up to:a b c Luo Y, Batalao A, Zhou H, Zhu L (February 1997). "Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system" (PDF). BioTechniques. 22(2): 350–2. doi:10.2144/97222pf02. PMID 9043710. Archived from the original (PDF) on 27 April 2017. Retrieved 26 April 2017.
    5. ^ Verschure PJ, Visser AE, Rots MG (2006). Step out of the groove: epigenetic gene control systems and engineered transcription factors. Advances in Genetics. 56. pp. 163–204. doi:10.1016/S0065-2660(06)56005-5. ISBN 9780120176564. PMID 16735158.[dead link]
    6. ^ Jump up to:a b Brückner A, Polge C, Lentze N, Auerbach D, Schlattner U (June 2009). "Yeast two-hybrid, a powerful tool for systems biology". International Journal of Molecular Sciences. 10(6): 2763–88. doi:10.3390/ijms10062763. PMC 2705515. PMID 19582228.
    7. ^ Gietz RD, Triggs-Raine B, Robbins A, Graham KC, Woods RA (July 1997). "Identification of proteins that interact with a protein of interest: applications of the yeast two-hybrid system". Molecular and Cellular Biochemistry. 172 (1–2): 67–79. doi:10.1023/A:1006859319926. PMID 9278233.
    8. ^ Jump up to:a b c d Auerbach D, Stagljar I (2005). "Yeast Two-Hybrid Protein-Protein Interaction Networks". Proteomics and Protein-Protein Interactions. Protein Reviews. 3. pp. 19–31. doi:10.1007/0-387-24532-4_2. ISBN 978-0-387-24531-7.
    9. ^ Whipple FW (August 1998). "Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli". Nucleic Acids Research. 26 (16): 3700–6. doi:10.1093/nar/26.16.3700. PMC 147751. PMID 9685485.


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