Protein-Protein Interaction

After sequencing the human genome, current research is focusing on the functional analysis of human gene products like proteins. The analysis of protein-protein interaction is important to identify the role of certain proteins in diseases and develop novel drug targets. The novel in vivo screening system Ras Recruitment System (RRS) offers additional possibilities to detect protein interactions as compared to the common yeast 2-hybrid system.

An automated screening system based on the MICROLAB STAR combined with a powerful database system is used for the detection of protein-protein interactions. The STAR is mainly used for hit-picking of yeast cultures from MTP´s after successful lead generation. An expansion of the system to include purification of proteins by filtration is also possible.

The screening system is capable of highly parallel interaction screenings of cDNA libraries. Candidates of interacting protein pairs are selected by RRS screening in living yeast cells, which form colonies on agar plates. Positive yeast colonies are picked from agar plates using a picking robot and transferred to culture medium in common MTP´s (master plates with barcode labels). These master plates are used to store the clones of interest as well as to perform specificity assays to test the possible interaction of the proteins.

The STAR has been integrated into a proprietary laboratory information management system (PIA-LIMS) which stores all interaction and specificity data and is used to identify interaction candidates with high confidence.

For hit-picking, the STAR obtains worklists online from the PIA-LIMS containing the location of positive samples in the MTP’s. The recognized positive hits are transferred to bar coded target MTP’s. In order to track samples, bar codes of the target plates and the new location of the samples are subsequently stored in PIA-LIMS. Finally the master plates are lidded and transferred back to the stacker.

The identity of the interacting proteins is revealed by sequencing the corresponding expression plasmids isolated from the positive yeast clones.
The generated protein interaction data will be used for data mining external genomic, proteomic and disease related databases and may lead to new applications in therapeutics and diagnostics as well.

Features and
Benefits

  • high precision in low volume pipetting
  • increased speed for the hit-picking process due to: independent spreading of the channels and the sequence-oriented software which is not restricted to processing only one plate at a time –allowing flexible hit-picking from several source plates simultaneously
  • semi-sterile environment thanks to the liquid-free pipetting system and the use of lids to cover plates
  • the STAR’s unique pipetting monitoring features (MAD and pLLD) increase process stability of optional vacuum-based protein purification steps
  • cost-efficient parallel use of reusable steel needles and disposable tips
  • parallel washing of steel needles increases the speed of the overall process

System
Configuration

  • MICROLAB® STAR, 4 channels with autoload option and iSWAP integrated plate handler
  • Wash Station with 3 module for 300µl Needles
  • BVS vacuum system nucleic acid clean up with different Kits (MN &Millipore)
  • H + P shaker
  • 4 Stackers on the left side of the instrument

Literature

  • Kruse C., S. Vasiliev, S. Hanke, H. Hennemann (2003).High-throughput interaction screening for the elucidation of protein kinase networks and the identification of protein kinase inhibitors. Cell Mol Biol Lett. 8: 585
  • Caesar – annual report 2002Kruse C., S. Vasiliev, S. Hanke, H. Hennemann (2002).Protein interaction analysis

Links

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