Team:Bielefeld-CeBiTec/Project/Selection/Bacterial Two-Hybrid System
Bacterial two-hybrid system
Motivation and Overview
It is utmost importance to our project to separate bacteria with high affinity binding proteins from bacteria with low or moderate affinity binding proteins. Therefore, we need an efficient selection system. One approach
is based on the concept of the very common yeast two-hybrid system, but optimized for bacterial selection directly in E. coli (
Theoretical background
The bacterial two-hybrid system is a possibility to separate cells from each other through differences in protein-protein interaction strength(
Our system in detail
The DNA binding domain
Our system is based on two fusion proteins. The first protein consists of our target protein fused with a DNA binding domain. In very early hybrid transcriptional activation system the frequently binding domains were different zinc finger proteins (Klug & Rhodes 1987). The zinc finger domain of the murein transcription factor Zif268 was most frequently used in two-hybrid systems. Three individual zinc finger motifs collectively bind a nine base-pair long sequence (Ramirez et al. 2008). Off-target effects were observed instead of a high affinity to the intended target. The revision of the zinc fingers make them on the one hand way more effective but also on the other hand way more complicated to use (Sander et al. 2011).
Therefore, DNA binding domains, which are a lot easier to use for bacterial two-hybrid systems are necessary. Examples are, the repressor proteins cI of the phages lambda (Hays et al. 2000), respectively. The protein family cI are repressor proteins that compete with the phage Cro proteins for DNA binding. The main function of them is the binding at the binding sites OR1 and OR2 on the DNA. If they bind at these sites they prevent that Cro can bind at the binding sites OR2 and OR3 and thereby inhibit the expression of the cro gene (
OR1 and OR2, also OL1 and OL2 downstream of the OR1 and OR2 sites are necessary for complete repression of the cro gene. Therefore, cI binds at the DNA as an octamere (Joung et al. 2000). All cI protein have specific binding sequences namely OR1 and OR2. These cI binding sequences are species specific and therefore different between diverse varying phages isolates. 
However, the design of a hybrid transcriptional activation system with cI as binding domain is easier than the use of a zinc finger protein. This is the due to the binding sequences for a specific cI, which does not require optimization like zinc fingers do.
Comparison of cI proteins to zinc finger proteins revealed a much higher expression rate of a reporter in a designed bacterial two hybrid system, if cI proteins is used as the DNA binding domain (
Therefore, DNA binding domains, which are a lot easier to use for bacterial two-hybrid systems are necessary. Examples are, the repressor proteins cI of the phages lambda (Hays et al. 2000), respectively. The protein family cI are repressor proteins that compete with the phage Cro proteins for DNA binding. The main function of them is the binding at the binding sites OR1 and OR2 on the DNA. If they bind at these sites they prevent that Cro can bind at the binding sites OR2 and OR3 and thereby inhibit the expression of the cro gene (
OR1 and OR2, also OL1 and OL2 downstream of the OR1 and OR2 sites are necessary for complete repression of the cro gene. Therefore, cI binds at the DNA as an octamere (Joung et al. 2000). All cI protein have specific binding sequences namely OR1 and OR2. These cI binding sequences are species specific and therefore different between diverse varying phages isolates. However, the design of a hybrid transcriptional activation system with cI as binding domain is easier than the use of a zinc finger protein. This is the due to the binding sequences for a specific cI, which does not require optimization like zinc fingers do.
Comparison of cI proteins to zinc finger proteins revealed a much higher expression rate of a reporter in a designed bacterial two hybrid system, if cI proteins is used as the DNA binding domain (
The activation domain
The second fusion protein of the hybrid transcriptional activation system is our Evobody fused to an activation domain. This domain is the impeller of the system. If the activation domain is recruited to the promoter through the interaction of the binding protein (the Evobody) and the DNA anchored target protein the gene expression of the reporter gene is strongly increased(Joung et al. 2000;Fu 2004, Ishihama 1992).
RpoZ, the much smaller omega subunit of the RNA polymerase, which could also be used as transcriptional activator. It is mainly important for the facilitation and stabilization of the assembly of the RNA polymerase (Badran et al. 2016). Therefore, we decided to use the RpoZ as activator domain.
Choice of the positive controls
Every system should be validate by analyzing a positive control. The two hybrid transcriptional activation system converts the binding affinity of two proteins in corresponding expression intensity. A well-documented protein-protein interaction is the binding of the regulator protein Gal4 of Saccharomyces cerevisiae with a modified version of the Gal11 regulatory protein Gal11P (Himmelfarb et al. 1990), which allows the binding of Gal11P in the dimerization domain of Gal4 (
In addition to the Gal4-Gal11P interaction a second positiv control should be tested. Having our library in mind we looked for a control that was very similar to our Evobodies. One protein with good structural similarities
is the antibody mimic or monobody HA4. (Strukturelle Ähnlichkeit zeigen) This monobody was designed by a working group in Chicago searching for an antibody mimic, that can bind the SH2 domain of the tyrosine kinase Abelson with low nanomolar affinity (Brender & Zhang 2015). In single mutation R38A in HA4 causes a reduction of transcriptional activation of the reporter gene of 35 to 40%. The mutation Y87A is even more detrimental leading to only 5 to 10% of the native binding affinity (
In addition to the Gal4-Gal11P interaction a second positiv control should be tested. Having our library in mind we looked for a control that was very similar to our Evobodies. One protein with good structural similarities
is the antibody mimic or monobody HA4. (Strukturelle Ähnlichkeit zeigen) This monobody was designed by a working group in Chicago searching for an antibody mimic, that can bind the SH2 domain of the tyrosine kinase Abelson with low nanomolar affinity (Brender & Zhang 2015). In single mutation R38A in HA4 causes a reduction of transcriptional activation of the reporter gene of 35 to 40%. The mutation Y87A is even more detrimental leading to only 5 to 10% of the native binding affinity (Design of the reporter
A selection system should be characterized based on different proteins with known mutual affinity values. A strong correlation between the binding affinity of the interacting proteins and the reporter gene activity is required. The selection of hogh affiniy Evobodies necessitates a reporter that confers growth advantage to the cells of interest. A good choice for such a reporter is an antibiotic resistance gene like bla against ampicillin (Roberts 1996), respectively. Increase of the antibiotic concentration would lead to a decrease of a bacterial population. Finally, the best Evobodies should allow the survival of the cells.
References
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