Using Dual Membrane Yeast Two Hybrid System to Elucidate TCR Signaling Components

1
Using Dual Membrane Yeast Two Hybrid System to
Elucidate TCR Signaling Components

• Safak Isil Nalbant ,PhD Student
• Sabanci University

2
Classical Yeast Two Hybrid

• Powerful method for the in vivo analysis of
  protein-protein interactions.
• Over 50 of interactions in scientific literature
  have been found by Y2H.
• But limited to soluble proteins and soluble
  domains of membrane proteins.
• An alternative Split-ubiquitin system.

3
Split-ubiquitin System

• Ubiquitin conserved 76 aa. protein.
• Attached to N-term. of proteins as a signal for
  degredation.
• Cleavage by Ubiquitin Specific Protease (UBP)

4
Split-ubiquitin System
5
Dual Membrane System
6
Dual Membrane System
Cell membrane
LexA
nucleus
Reporter transcription
HIS3,ADE2,lacZ
7
Project

• Using dual membrane systems show any interaction
  between TCR complex subunits.
• Screening Jurkat T cell cDNA library detect any
  possible interacting protein with any of the TCR
  subunits?.

8
First part of the project

• Cloning TCR subunits into the bait vector.
• Testing growth of NMY51 yeast cells on minimal
  plates( both selective and non-selective)
• Transformation of the bait constructs into NMY51.
• Verifying expression of bait using the control
  assay.
• Optimizing the screening stringency using a pilot
  screen.

9
Amplification of TCR Subunits
TCRa
TCRb
CD3d
CD3e
CD3g
pTCRa
Figure 1 Amplification of TCR subunits via PCR
10
Cloning of TCR subunits into bait and prey
vectors

• PCR amplified subunits are cloned into bait
  vectors (and also prey vectors) using Sfi1 sites
  in the vectors.

Prey Vector
Bait Vector
Sfi1
PCR
11
Growth of NMY51 Yeast Cells

• No visible growth on leu,-trp,-his,-ade,-ura
  media.
• When transformed with bait vector expressing LEU2
  grows on leu plates, when transformed with prey
  vector expressing TRP1 grows on trp plates.

Figure2 Bait expressing NMY51
Figure3 Prey expressing NMY51
Figure4 NMY51 on selective plates
12
Control Assay-Positive
13
Control Assay-Negative
14
Results of Control Assay
Selective Plates
Non-Selective Plates
Figure 5 Bait-cd3d expressing NMY51 transformed
with positive control prey
15
Results of Control Assay
Positive Control
Negative Control
Figure 6 Bait-cd3d expressing NMY51 transformed
with positive and negative control prey
16
Growth under selection
growth under selection 75
17
What to do next?

• Optimizing the screening stringency using a pilot
  screen. (ongoing)
• Verifying expression of bait by Western blot
  (using a LexA)and flow cytometric analysis.
• Library transformation and selection of
  interactors.
• Assays for detection of b-galactosidase activity.
• Confirmation and characterization of positive
  interactors.(long-term)

18
Acknowledgments

• Batu Erman Lab ?
• Tübitak (project 104T237)

19
ANY QUESTIONS
?