PROTEIN MICROARRAY AND EXPRESSION CORE

 

Protein Microarray and Expression Core

Phillip Felgner, University of California, Irvine

Abstract:
Despite the ever increasing availability of genome sequences from many human pathogens the production of complete proteomes remains a bottleneck. To address this, a high throughput PCR recombination cloning and expression platform has been developed. The method relies on high throughput amplification of each predicted open reading frame using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are then expressed in an E. coli-based cell-free in vitro transcription/translation system and the proteins are printed directly onto microarrays without further purification. The chips can then be used to screen sera from individuals naturally or experimentally exposed to different infectious microorganisms. With support from 3 NIH/NIAID biodefens grants, the laboratory has completed the proteomes for vaccinia virus (194 ORFs) and for F. tularensis (1933 ORFs), and has initiated synthesis of the B. pseudomallei proteome (~5600 ORFs). The results of serology using these chips from vaccinated or infected, animals and humans are being applied by this laboratory to the problems of vaccine and diagnostic antigen discovery. Numerous inter- and intra-RCE collaborative projects have been initiated. The services, and inventory of vectors and proteins will advertised and accessible through an on-line Web site that is in preparation.