Mcw Research Foundation, Inc.

Arthur L Haas

Joseph T Barbieri

Brian D Halligan

Lawrence M Ryan

Daniel J McCarty

Jana Narasimhan

Ikuko Masuda

We have cloned and sequenced the cDNA encoding the 61 kD active fragment of a unique porcine chondrocyte nucleotide pyrophosphohydrolase (NTPPHase) from a porcine chondrocyte library. Degenerate oligonucleotides, corresponding to the N-terminal amino acid sequence of this peptide were hybridized to porcine chondrocyte cDNA and used to amplify DNA encoding the N-terminal sequence of 61 kD with the polymerase chain reaction (PCR). The PCR products were then used as probes to clone the entire open reading-frame for the 61 kD fragment from a porcine chondrocyte cDNA library. The length of the cloned cDNA was 2509 bp. Translation of the open-reading-frame predicts the 61 kD fragment to be a 459 amino acid protein. BLAST and FASTA analysis confirmed that this amino acid sequence was unique and did not possess high homology to any known proteins in the non-redundant protein data base. Limited homology (17%) between the 61 kD fragment and several prokaryotic and eukaryotic ATP pyrophosphate-lyase (adenylate cyclase) was detected. Northern blot analysis of porcine chondrocyte RNA showed that the DNA encoding the 61 kD fragment hybridized to a 4.3 kbp RNA transcript. Human chondrocyte RNA also hybridized to this porcine DNA probe. Coupled in vitro transcription translation of an expression vector containing the DNA insert in frame showed the expression of a 61 kD protein.

Cloned nucleotide pyrophosphohydrolase and uses thereof

A method of preparing recombinant ubiquitin cross-reactive protein (UCRP) is disclosed. In one embodiment, the method comprises the step of purifying UCRP from a host cell in the presence of a cobalt or Group IIb metal, wherein the cobalt or Group IIb metal prevents proteolytic inactivation of UCRP occurring by cleavage of the UCRP carboxyl terminal glycine dipeptide. In another embodiment the method comprises purifying UCRP comprising an additional arginine or lysine residue at the carboxy terminal. A preparation of UCRP with an additional arginine residue at the carboxy terminus is also disclosed.

Preparation of recombinant ubiquitin cross-reactive protein (UCRP) with

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Brookfield, WI, United States of America

Arthur L Haas