Characterization of novel Proteins by Analysis of Human Protein Networks:
Part1 - cloning of human full ORFs of novel proteins

Bernhard Korn
RZPD - Ressourcenzentrum für Genomforschung
INF 506
69120 Heidelberg
Germany
telephone: +49 (6221) 42 4700
fax: +49 (6221) 42 4704
email: korn@rzpd.de
prestype: Platform
presenter: Korn

R. Schatten¹, S. Henze¹, M. Schick¹, S. Wiemann², A. Gödde¹, H. Göhler³, E. Wanker³, B. Korn¹

1 RZPD ­ Ressourcenzentrum für Genomforschung, INF 506, 69120 Heidelberg, Germany
2 Deutsches Krebsforschungszentrum, INF 506, 69120 Heidelberg, Germany
3 Max-Planck Institut für Molekulare Genetik, Ihnestr. 73, 14195 Berlin, Germany

The sequencing of the human genome has resulted in the identification of a large number of novel proteins, whose function and interactions with other proteins have to be determined. We aim at the characterization of unknown proteins and the assignment of these proteins to particular pathways or multimeric structures. We apply a systematic, high throughput approach for functional analysis. We have developed an automated, medium throughput cloning and verification strategy that allows us to clone human full ORF cDNAs at a rate of 100 per week. Up to now, we have cloned and completely sequence verified more than 2.400 ORFs. We plan to clone 6.000 ORFs within the next year.

These ORFs are so far not characterised on the functional level, except for computational predictions, many have been identified by the German cDNA Consortium. It is our intention to channel these clones to a medium size yeast two-hybrid system that allows the screening for protein-protein interactions. Within our current project, up to 5,000 defined human cDNAs will be inserted into a DNA binding domain vector for screens against different human cDNA libraries, cloned into activation domain vectors.

Special emphasis will be put on protein interactions with medically relevant proteins such as oncology related proteins, proteins involved in cardiovascular defects, and in neurodegenerative diseases. Protein-protein interactions will be confirmed by in vitro binding experiments, co-immunoprecipitations and co-localisation studies. Finally, the results of the two-hybrid screens and of the functional characterisations will be stored in the Primary Database at RZPD. With this novel systematic approach we will combine for the first time high quality sequence information with large-scale functional data in a common database. We suggest that this information will add value to newly identified genes and our overall understanding of protein networks. Finally, clones will be made available for public use via the RZPD.