Part:BBa_K2082224

Double mutated fusion protein HA4(R38A,E52A):RpoZ generator

HA4(R38A,E52A):RpoZ fusion protein BBa_K2082202

This BioBrick is a fusion protein containing the double mutated monobody HA4(R38A,E52A) and the omega subunit of the RNA polymerase I (RpoZ) of E. coli. The mutated HA4:RpoZ proteins Y87A (BBa_2082202), R36A (BBa_2082203) and the double mutated protein R38A_E52A (BBa_2082204) are designed to the results of Bedran et al. (2016). The mutations in HA4 leads to a decrease of the binding affinity to the interaction partner SH2. In consideration of the bacterial two-hybrid system, a lower binding affinity consults in a lower transcriptional activation of the reporter gene. Therefore, these controls allow a detail characterization of a bacterial two-hybrid system using HA4 and SH2 as positive control.

Figure 1: Transcriptional activity by using different mutated HA4 proteins. Illustrated is the percental transcription activity dependent on the binding affinity to the SH2 domain.

This is the generator of the BioBrick BBa_2082204.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 166
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The design of the HA4:RpoZ fusion protein was made with a specific cMyc-linker between the two compartments. This linker was designed to create a possibility of a direct evidence for the expression with the use of an anti-cMyc antibody. To confirm that the anti-myc antibody binds correctly to the HA4 Evobody, a bio-layer interferometry was performed with a BLItz system by Forté Bio. The HA4 monobody was immobilized on an amine reactive 2nd generation biosensor by Forté Bio. As the secondary supplement, the anti-cMyc antibody was used. As it can be seen in Figure 1, after the addition of the anti-myc antibody a wavelength shift occured, which shows that an association of the anti-myc antibody to the HA4 Evobody happened. When a regeneration solution was added, a dissociation of the anti-myc antibody could be observed. Therefore, the proof was given that the anti-cMyc antibody is able to interact with the designed cMyc-linker.

Figure 1: BLItz results of immobilized HA4 with anti-myc antibody: Result of a bio-layer interferometry of HA4 with anti-myc antibody on Amine reactive 2nd generation biosensor.

By blotting a SDS-gel containing the samples of the HA4:RpoZ generator BBa_2082221 and the mutated HA4:RpoZ generators BBa_2082222, BBa_2082223 and BBa_2082224 a standing out band was seen on every lane in the gel which was emphasized to the visible background noise. Through the wandering speed of the protein in the gel and in comparison with the plotted ladder the detected proteins had a mass of about 24 kDa. These results confirm the expected mass of the fusion protein HA4:RpoZ with 24.14 kDa. Therefore, an expression of the fusion proteins are confirmed.

Figure 2. Western blot for expression control: Result of a western blot with samples from left to right: marker, HA4 Evobody wild type, HA4 Y87A mutant Evobody, HA4 R38A mutant Evobody, HA4 R38A E52A, SH2:cI+HA4 Evobody