Metalloproteases of the neprilysin family

Details Metalloproteases of the neprilysin family Metalloproteases of the neprilysin family

2006-07-04

2006-07-04

Rao, Manjunath (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S004000, C435S006120, C435S252300, C435S320100, C435S069100, C435S071100, C435S440000, C435S024000, C536S023200, C536S023500

Reexamination Certificate

active

07070974

ABSTRACT:
In this paper, we describe RT-PCR strategies that allowed us to identify and clone members of the NEP-like family. Degenerate oligoncleotide primers corresponding to consensus sequences located on either side of the HEXXH consensus sequence for zincins were designed and used in RT-PCR with mouse and human testis cDNAs. DNA fragments with lengths expected from the sequence of this class of enzympes were obtained. These DNA fragments were cloned and sequenced. Using this PCR strategy and the PCR fragments as probes to screen cDNA libraries, three zincin-like peptidases were identified in addition of known members of the family. The cDNA sequences allowed to derive specific probes for Northern and in situ hybridization, and probe human chromosomes to localize the gene and establish potential links to genetic diseases. Furthermore, these cDNA sequences were used to produce recombinant fusion proteins inEscherichia coliin order to raise specific antibodies. Finally, the cDNA sequences were cloned in mammalian expression vectors and transfected in various mammalian cell lines to produce active recombinant enzymes suitable for testing specific inhibitors.

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