Creative Biomolecules, Inc.

Genentech, Inc.

Micromet Ag

A. Menarini S.a.s.

Roberto Crea

Charles M Cohen

Hermann Oppermann

Peter C Keck

Marc F Charette

David C Rueger

James S Huston

Richard J Ridge

David V Goeddel

Roy H Pang

Gay-May Wu

Gabriel Alvarado-Urbina

Disclosed is a method for the isolation and purification of polypeptides expressed in host cells by recombinant DNA techniques. A fused polypeptide is produced containing a desired polypeptide fused to additional amino acids. The additional amino acids define a leader sequence having properties exploitable in purification, a hinge region, and a cleavage site. The hinge region is cysteine-free and has a secondary structure which serves to expose the cleavage site to a selected endopeptidase. The method of the invention involves the production of a fused polypeptide which may be efficiently isolated by exploiting the properties of the leader sequence, and then efficiently cleaved at the cleavage site in an appropriate aqueous environment by virtue of the influence of the hinge on the cleavage agent/cleavage site reaction and other properties of the fused polypeptide.

Product and process for the production, isolation, and purification of recombinant polypeptide

Protein analogues of tissue plasminogen activator (tPA) are described. The analogues exhibit the proteolytic function of natural tPA and optionally fibrin binding activity, but are molecules, generally of lower molecular weight than natural tPA, designed for efficient expression in prokaryotic host cell systems. The analogues can comprise a catalytic fragment of tPA or a catalytic fragment of tPA linked to a polypeptide which stabilizes the catalytic fragment, provides for efficient expression of the fragment or confers a fibrin binding capability. Fibrin binding polypeptides can be a polypeptide fragments derived from tPA which embody the fibrin binding domain(s) of natural tPA or they can be an exogenous (non-tPA) polypeptides of eukaryotic or prokaryotic origin which exhibit fibrin binding affinity such as the antigen binding fragment of an antifibrin immunoglobulin or the B domain of protein A. Genetic constructs for expression of the analogues are also provided.

Protein analogues of tissue plasminogen activator

A cDNA library is constructed using mRNA from human fibroblasts induced with poly(I):poly(C). A bacterial clone containing fibroblast interferon cDNA sequences identified by hybridization to a cDNA probe synthesized using deoxyoligonucleotide primers which hybridize to fibroblast interferon mRNA specifically. Expression plasmids are constructed which permit the synthesis in E. coli of 8.times.10.sup.7 units of human fibroblast interferon per liter of culture. The bacterially produced fibroblast interferon is indistinguishable from authentic human fibroblast interferon by several critieria.

Mature human fibroblast interferon

Disclosed are methods for increasing the yield of intact target proteins by cleaving fused polypeptides made by recombinant DNA techniques. The fused polypeptides are designed at the DNA level to have a preselected primary cleavage site in a pendant polypeptide fused to a protein of interest. Structural features of the fused polypeptide and cleavage reaction environment are controlled to favor cleavage by a preselected cleavage agent at the primary cleavage site over a second cleavage agent-sensitive amino acid sequence in the target protein. The cleavage reaction is terminated before completion when the ratio of intact target protein to truncated, cleaved target protein is optimized, and the remaining reaction mixture comprising uncleaved fused polypeptide is resubjected to the cleavage agent. The presence of charged organic molecules in the cleavage reaction mixture favors cleavage at the primary cleavage site. The endopeptidase used for cleavage may be immobilized on an insoluble support matrix.

Enhanced proteolytic cleavage of recombinant fusion proteins

Product and process for introduction of a hinge region into a fusion

Disclosed is a process for producing a fusion protein of epidermal growth factor (EGF) attached through a Glu-residue, treatment of the fusion protein with a Staphylococcus aureus protease specific for cleaving peptides at a Glu-linkage thereby producing large amounts of the 53 amino acid EGF sequence. The treatment conditions optimize production of large amounts of the 53 amino acid sequence relative to the 51 amino acid sequence minus the 52 Leu and 53 Arg residues and take full advantage of the selectivity of the protease.

Process for producing human epidermal growth factor and analogs thereof

Methods and compositions are provided for the production of gene mutations and protein analogs using synthetic gene design and double-stranded synthetic oligonucleotide cassettes. The disclosure provides synthetic structural genes, synthetic double-stranded oligonucleotide cassettes and methods for the creation of synthetic gene analogs and polypeptide analogs by shortening or altering the native amino acid sequence.

Production of gene and protein analogs through synthetic gene design

Disclosed is a process for producing a fusion protein comprising epidermal growth factor (EGF) attached through a Glu-residue to a leader. Treatment of the fusion protein with a Staphylococcus aureus V8 protease specific for cleaving peptides at a Glu-linkage produces large amounts of the 53 amino acid EGF sequence. The treatment conditions optimize production of the 53 and 51 amino acid EGFs relative to shorter analogs, and take full advantage of the selectivity of the protease.

Compounds of the formula ##STR1## where R is a base labile substituted-phenyloxy protecting group, are useful as phosphorylating reagents in the synthesis of oligonucleotides by a phosphotriester method.

Nucleosidic phosphorylating agent and methods

Compounds of the formula ##STR1## where R is a base labile substituted-phenyloxy protecting group, are useful as phosphorylating reagents in the synthesis of oligonucleotides by a phosphotriester method. Such compounds are made by the reaction of .beta.-cyanoethanol and a corresponding R-phosphorodichloridate. A method of phosphorylating involves reacting such compounds, in the presence of base, with a nucleoside or a polynucleotide having a free 3' hydroxyl group, having the 5'--0 position protected by an organic group labile in acidic medium and having any phosphotriester linking groups protected by an organic group labile in strong basic medium.

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Boston, MA, United States of America

Roberto Crea