University System of Maryland

Genex Corporation

Other

Enzon, Inc.

Enzon Labs Inc.

Philip N Bryan

Michael W Pantoliano

Patrick A Alexander

Susan L Strausberg

Michele L Rollence

Barry C Finzel

Chi H Wong

The present invention is directed to the identification of a protease prodomain that is capable of binding a corresponding protease with high affinity. The protease prodomain of the present invention is fused to a second protein to form a protease prodomain fusion protein. The presence of a protease prodomain protein in a fusion protein allows for easy and selective purification of the second protein by incubation with the corresponding protease.

Engineered proteases for affinity purification and processing of fusion proteins

Novel calcium free mutants are taught, in particular which have been mutated to eliminate amino acids 75-83 and which retain enzymatic activity and stability. Recombinant methods for producing same and recombinant DNA encoding for such mutants are also provided.

Calcium free subtilisin mutants

Novel calcium free subtilisin mutants are taught, in particular subtilisins which have been mutated to eliminate amino acids 75-83 and part or all of amino acids 1-22 (the N-terminal region) and which retain enzymatic activity and stability. Recombinant methods for producing the same and recombinant DNA encoding for such subtilisin mutants are also provided.

Novel calcium free subtilisin mutants are taught, in particular a subtilisin which has been mutated to eliminate amino acids 75-83.

Novel calcium free subtilisin mutants are taught, in particular subtilisins which have been mutated to eliminate amino acids 75-83 and which retain enzymatic activity and stability. Recombinant methods for producing same and recombinant DNA encoding for such subtilisin mutants are also provided.

Subtilisin mutants lacking a primary calcium binding site

The present invention provides a method for redesigning proteins to increase the stability of the protein by altering amino acid residue(s) that are in close proximity to the protein's metal ion binding site(s). Further, the invention is directed to proteins that have been redesigned to have enhanced stability according to the methods of this invention.

Engineering of electrostatic interactions at metal ion binding sites for

The invention relates to subtilisin enzymes which have been modified by mutating a nucleotide sequence (gene) coding for the subtilisin. The modified subtilisin enzymes have enhanced thermal stability.

Thermally stable serine proteases

The invention relates to the novel use of mutants of subtilisin in organic syntheses reactions in non-native environments. Especially the invention relates to methods for the use of mutant subtilisins in organic solvents for the catalysis of reactions involving ester formation and cleavage, including acylations and deacylations, and amidations and deamidations. The methods provide novel strategies which are useful in the synthesis of deoxynucleosides, dideoxynucleosides, peptides, sugars and the like.

Biosynthetic uses of thermostable proteases

Subtilisin mutations

The invention relates to modified subtilisin enzymes which have increased thermal stability. The modified subtilisin enzymes have at least two or more amino acid mutations which confer increased thermal stability. It has been discovered that combining individual stabilizing mutations in subtilisin frequently results in an additive increase in thermal stability. In addition, the invention pertains to cloned mutant genes coding for a subtilisin material having at least two amino acid substitution which has increased thermal stability.

Combining mutations for stabilization of subtilisin

Cloned DNA is mutated by creating a single-stranded target region in a cloned DNA segment, and introducing a mutation into the single-stranded target region by treating the target region with a chemical or biological mutagenizing agent capable of introducing mutations into single-stranded DNA. The mutated target region then is rendered double-stranded and a microorganism is transformed with the mutated double-stranded DNA present in an expression vector. The transformed microorganism is cultivated under conditions wherein the mutated DNA is expressed to form an expression product, and the expression product is screened to identify a desired mutation in the DNA segment. Mutant subtilisins of enhanced thermal stability are also disclosed.

Growing community of inventors

Silver Spring, MD, United States of America

Philip N Bryan