Compartmentalized linkage of genes encoding interacting protein pairs*
Colour Online: See the article online to view all figures in colour.
Abstract
Emulsion technology has been successfully applied to the fields of next‐generation high‐throughput sequencing, protein engineering and clinical diagnostics. Here, we extend its scope to proteomics research by developing and characterizing a method, termed iCLIP (in vitro compartmentalized linkage of interacting partners), which enables genes encoding interacting protein pairs to be linked in a single segment of DNA. This will facilitate archiving of the interactomes from library versus library two‐hybrid screens as libraries of linked DNAs. We further demonstrate the ability to interrogate a model yeast two‐hybrid iCLIP library for interactants by “PCR‐pulldown,” using a primer specific to a gene of interest along with a universal primer. iCLIP libraries may also be subjected to high‐throughput sequencing to generate interactome information. The applicability of the technique is also demonstrated in the related context of the bacterial two‐hybrid system.
Number of times cited: 4
- Nozomu Yachie, Evangelia Petsalaki, Joseph C Mellor, Jochen Weile, Yves Jacob, Marta Verby, Sedide B Ozturk, Siyang Li, Atina G Cote, Roberto Mosca, Jennifer J Knapp, Minjeong Ko, Analyn Yu, Marinella Gebbia, Nidhi Sahni, Song Yi, Tanya Tyagi, Dayag Sheykhkarimli, Jonathan F Roth, Cassandra Wong, Louai Musa, Jamie Snider, Yi‐Chun Liu, Haiyuan Yu, Pascal Braun, Igor Stagljar, Tong Hao, Michael A Calderwood, Laurence Pelletier, Patrick Aloy, David E Hill, Marc Vidal and Frederick P Roth, Pooled‐matrix protein interaction screens using Barcode Fusion Genetics, Molecular Systems Biology, 12, 4, (2016).
- Uri Laserson, Francois Vigneault, Daniel Gadala-Maria, Gur Yaari, Mohamed Uduman, Jason A. Vander Heiden, William Kelton, Sang Taek Jung, Yi Liu, Jonathan Laserson, Raj Chari, Je-Hyuk Lee, Ido Bachelet, Brendan Hickey, Erez Lieberman-Aiden, Bozena Hanczaruk, Birgitte B. Simen, Michael Egholm, Daphne Koller, George Georgiou, Steven H. Kleinstein and George M. Church, High-resolution antibody dynamics of vaccine-induced immune responses, Proceedings of the National Academy of Sciences, 111, 13, (4928), (2014).
- Takeshi Sumida, Hiroshi Yanagawa and Nobuhide Doi, In VitroSelection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR, Journal of Nucleic Acids, 2012, (1), (2012).
- Haiyuan Yu, Leah Tardivo, Stanley Tam, Evan Weiner, Fana Gebreab, Changyu Fan, Nenad Svrzikapa, Tomoko Hirozane-Kishikawa, Edward Rietman, Xinping Yang, Julie Sahalie, Kourosh Salehi-Ashtiani, Tong Hao, Michael E Cusick, David E Hill, Frederick P Roth, Pascal Braun and Marc Vidal, Next-generation sequencing to generate interactome datasets, Nature Methods, 8, 6, (478), (2011).
