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A Novel Screen Identifying Ternary Binding Partners Of Know Interacting Proteins

Ref-No: TA-EKUT-13-0304 C.Grefen


Abstract

Here, we present the first screening system in yeast that permits high-throughput screening of cDNA libraries for ternary binding partners of a known interaction couple. This is achieved by a unique combination of SUB and yeast mating. Technical prerequisites are special “2in1”-vectors, which allow simultaneous transformation of “Bait I” and “Bait II” on a single plasmid in yeast of one mating type and the cDNA library in the other.


Background

Many biological processes depend on oligomeric protein-protein interactions (PPI). However, state-of-the-art in vivo PPI techniques focus on analysing binary interactions (i.e. the Split-Ubiquitin System (SUS)1). The SUS can also be used to analyse binding of three proteins in the so-called SUS Bridge Assay (SUB)2 . Nevertheless, this assay makes an unbiased screening approach cumbersome and its low efficiency restricts the identification of meaningful candidates.


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Problem

The search for potential, yet unknown, third protein binding partners (to two already known proteins) within a given cDNA library by using the SUS Bridge Assay (SUB) requires a rather cumbersome cloning protocol step by step for each given cDNA-clone (transformation of individual plasmids for each assumed cDNA element). In the day-to-day routine of labs this is only feasible for a few preselected proteins, not for a screening of a whole cDNA library.

 

 


Solution

To enable the screening of a whole cDNA-library we combine the genes for two already known Bait-proteins of a SUS (Bait I and Bait II) in a new 2in1-vector. Transformation of a so called Mat a-yeast strain with this 2in1 vector renders the possibility of using another complementary yeast-strain, a so-called Mat alpha strain, as a vehicle for a whole cDNA library. The scientist can transform any given cDNA library into this second yeast strain. Thus a yeast mating based approach becomes possible. After mating of Mat a and Mat alpha, yeast clones that are expressing protein complexes with at least three interacting and binding partners will render a positive signal.

The mating-based approach is facilitated through simultaneous cloning of both bait proteins on one plasmid backbone and the cDNA library on a second; each of which is either transformed in mating type a or alpha haploid yeast cells, respectively. As proof of concept we have identified novel interaction partners of the brassinosteroid receptor complex of Arabidopsis thaliana.

 

 


Advantages

- Easy identification of still unknown interacting protein binding partners to two already known proteins, this becomes part of the daily routine in laboratories

- Reliable: the new screen identifies real trimeric interaction partners as verified by our data

- Efficient: the mating-based approach guarantees 10-100 times higher coverage of primary interactions compared to standard approaches

- Low-cost: libraries can be re-used and stored in yeast, ready-to-use

 

 


Scope of application

- cDNA libraries from virtually all species can be tested

- The system can be expanded to identify quarternary interactions

 

 

 

 

 


Service

License or sale for commercial use

collaboration for further development


Eberhard Karls Universität Tübingen

Dr. Rolf Hecker
+49 (0)7071-2972639
r.hecker@uni-tuebingen.de
www.technologietransfer.uni-tuebingen.de
Address
Keplerstraße 2
72074 Tübingen



Development status

Prototype


Patent situation

  • DE pending
  • PCT pending

Keywords

protein-protein binding, Split-Ubiquitin-System (SUS), Screening System, cDNA-library, protein interaction, trimeric binding assay, 2in1 vector

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