The Johns Hopkins University

Ilse I Blumentals

Stephen H Brown

Robert M Kelly

Christian B Anfinsen

Anne K Robinson

Deb K Chatterjee

Mary C Longo

A John Hughes, Jr

Brian J Schmidt

Roger S Lasken

The invention relates to a substantially pure thermostable DNA polymerase. Preferably, the DNA polymerase has a molecular weight of about 95 kilodaltons and is more thermostable than Taq DNA polymerase. The present invention also relates to cloning and expression of the DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes.

Thermophilic DNA polymerase

Cell-free extracts from Pyrococcus furiosus were found to possess unusually high levels of proteolytic activity as measured by hydrolysis of azocasein; loss in activity was only 30% after incubation for 24 hours at 98.degree. C. and the half-life of proteolytic activity at that temperature was about 60 hours. Furthermore, cell-free extracts incubated at 98.degree. C. in 1% sodium dodecyl sulfate (SDS) for 24 hours yielded an SDS-resistant protease having a temperature optimum of at least 100.degree. C. The enzyme retained at least 40% of its activity when tested at 98.degree. C. by azocasein hydrolysis in the presence of 4M urea, 2M guanidinium chloride, 10 mM dithiothreitol or 150 mM .beta.-mercaptoethanol. The protease was found to have a pH optimum of 6.8 at 98.degree. C. and retained more than 45% of its activity at pH 9.3 and 82% of its activity at pH 4.5 in assays performed at those values. The protease was classified as a metalloprotease through inhibitor studies, and peptide hydrolysis showed trypsin-like cleavage with additional activities.

Proteolytic enzymes from hyperthermophilic bacteria and processes for

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Ilse I Blumentals