Cornell Research Foundation Inc.

David B Wilson

Michael L Shuler

Larry P Walker

John E Kinsella

Sheng Zhang

Todd W Gusek

Guifang Lao

A single celled organism, vector, and method are provided for continuous production of excreted proteins in the absence of substantial host cell replication and without the necessity for the addition of antibiotics to control cell replication. The vector has (1) an inducible promoter capable of activating a gene for a protein to be produced under conditions that substantially inhibit host cell replication, and (2) a hybrid gene containing a signal sequence fused to a gene for the protein to be produced. Large quantities of the transformed single celled organisms containing the vector can be grown in the absence of inducing conditions, thereby reducing the problem of plasmid loss. Further, the protein is produced under conditions that substantially inhibit host cell replication, thereby allowing immobilization of the single celled organism by entrapment or attachment within or onto a solid support surface. The single celled organism can be immobilized after, before or simultaneously with induction. The single celled organism advantageously may be an encapsulated gram-negative bacterium which has no outer membrane or an outer membrane that is permeable to the proteins to be produced so that the protein products are excreted into the media thereby avoiding difficulty in recovering the desired protein products due to the formation of inclusion bodies or the presence of contaminants produced by cell lyses.

System useful for the production of proteins from recombinant DNA in

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

Nucleotide sequences encoding a thermostable alkaline protease

The invention relates to a gene isolated from Thermomonospora fusca, wherein the gene encodes a thermostable cellulase. Disclosed is the nucleotide sequence of the T. fusca gene; and nucleic acid molecules comprising the gene, or a fragment of the gene, that can be used to recombinantly express the cellulase or a catalytically active polypeptide thereof, respectively. The isolated and purified recombinant cellulase or catalytically active polypeptide may be used to hydrolyze substrate either by itself; or in combination with other cellulases, with the resultant combination having unexpected hydrolytic activity.

Thermostable cellulase from a thermomonospora gene

An exocellular protease from Thermomonospora fusca YX and a process for producing the protease which has the following physicochemical properties: (1) Molecular mass: The protease has a molecular mass of from about 10,000 to 14,000 Daltons as measured by SDS-polyacrylamide gel electrophoresis; (2) Influence of inhibitors: The protease activity is inhibited by serine protease inhibitors; (3) Substrate specificity: A non-specific protease which can hydrolyze food proteins and bovine serum albumin at a rate of 50-100 nmoles peptide bonds/.mu.g enzyme/minute at 55.degree., pH 8.5 in 0.5 M Tris buffer without showing any substrate and/or product inhibition; (4) Reactivity: A broad spectrum serine type protease having activity at least 5 times greater than trypsin or chymotrypsin towards food grade proteins and bovine serum albumin; (5) Optimum activity temperature and temperature range: The optimum activity temperature is 80.degree. C. at pH of 8.0 in 0.05 M Tris buffer at an ionic strength of 0.2 M NaCl; The temperature range is 35.degree. to 95.degree. C. under the same pH, buffer, and ionic strength; (6) pH range and optimum pH value: The protease has a pH activity range of from about 7 to 11, and the optimum pH value is 9.0; (7) Tolerance to ionic strength conditions and optimum ionic strength: The protease is tolerant to ionic strengths of from 0.0 to about 1.0 M NaCl; Optimum ionic strength is 0.2 M NaCl; (8) Isoelectric point: The isoelectric point is at an alkaline pH; and (9) Structure: The protease is a monomer.

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Ithaca, NY, United States of America

David B Wilson