Detecting Membrane Protein-protein Interactions Using the Mammalian Membrane Two-hybrid (MaMTH) Assay

2017 ◽  
Vol 9 (1) ◽  
pp. 38-54 ◽  
Author(s):  
Punit Saraon ◽  
Ingrid Grozavu ◽  
Sang Hyun Lim ◽  
Jamie Snider ◽  
Zhong Yao ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Two Hybrid

scholarly journals Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

2009 ◽  
Vol 284 (24) ◽  
pp. 16369-16376 ◽  
Author(s):  
Xuebo Hu ◽  
Sungkwon Kang ◽  
Xiaoyue Chen ◽  
Charles B. Shoemaker ◽  
Moonsoo M. Jin
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Coiled Coil ◽  
Affinity Maturation ◽  
Antibody Binding ◽  
Soluble Form ◽  
Protein Protein Interactions ◽  
Yeast Surface ◽  
Two Hybrid

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

scholarly journals A Field-Proven Yeast Two-Hybrid Protocol Used to Identify Coronavirus–Host Protein–Protein Interactions

2015 ◽  
pp. 213-229 ◽  
Author(s):  
Pierre-Olivier Vidalain ◽  
Yves Jacob ◽  
Marne C. Hagemeijer ◽  
Louis M. Jones ◽  
Grégory Neveu ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Host Protein ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Two Hybrid

scholarly journals Analysis of Vaccinia Virus−Host Protein−Protein Interactions: Validations of Yeast Two-Hybrid Screenings

2009 ◽  
Vol 8 (9) ◽  
pp. 4311-4318 ◽  
Author(s):  
Leiliang Zhang ◽  
Nancy Y. Villa ◽  
Masmudur M. Rahman ◽  
Sherin Smallwood ◽  
Donna Shattuck ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Protein Interactions ◽  
Host Protein ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Two Hybrid
Sign in / Sign up

Export Citation Format

Share Document