Thermogen Inc.

Other

Malcolm J Casadaban

David C Demirjian

Veronika Vonstein

J Mark Weber

Nikos C Pagratis

Michael Fonstein

John Aikens

Larry Allen

George L Gaines

David DeMirjian

We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including ., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kan ). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu Km transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.

Recombinant DNA and methods for producing thermostable enzymes

The instant invention encompasses isolated stable esterase enzymes characterized by the ability to remain stable at certain temperatures, substrate specificities, and activity profile.

Stable biocatalysts for ester hydrolysis

The instant invention encompasses isolated stable esterase enzymes characterized by the ability to remain stable at certain temperatures, substrate specificities, and activity profile; the expression vectors which can express, nucleic acids which encode for, and corresponding protein amino acid sequence of such proteins.

The instant disclosure teaches transposons and methods of the instant invention for making Protein Fusions by rapid, random shuffling of protein domains to produce novel protein fusions. This system is generally applicable to production of multifunctional chimeric proteins in vivo and in vitro. The methods and constructs of the instant invention can be used to randomly create both carboxy- and amino-terminal protein fusions in vivo. The methods and contructs of the instant invention are useful in the development of a protein domain library, in the construction of multifunctional enzymes, and in the accelerated evolution of new enzymatic activities.

Protein fusion method and constructs

We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including Thermus flavus., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kan.sup.tr2). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu.sup.- Km.sup.r transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.

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Chicago, IL, United States of America

Malcolm J Casadaban