Bionebraska, Inc.

University of Nebraska

Restoragen, Inc.

Other

Nps Pharmaceuticals, Inc.

Carlsberg A/s

Medtronic, Inc.

Fred W Wagner

Jay S Stout

Dwane E Wylie

Sheldon M Schuster

Thomas R Coolidge

Bruce E Partridge

Dennis B Henriksen

Yuannan Xia

William Lewis

Peng Luan

Barton Holmquist

Daniel J Strydom

Gino Van Heeke

Bart Holmquist

Jin Seog Seo

Shane Manning

Klaus Breddam

Julie A Frank

Mary J Bossard

Morten C Kielland-Brandt

Edwin H Merrifield

Kjeld Ove Olesen

Uffe Hasbo Mortensen

Henning Ralf Stennicke

Randall R Carlson

Malcolm Riddell

Robert R Recker

John P Markwell

Osvaldo Lopez

Mark K Gutniak

H Edward Grotjan

Julie Heinrich

Dan Dormady

The invention provides methods for making peptides from a polypeptide containing at least one copy of the peptide using clostripain to excise the peptide from the polypeptide. The methods enable the use of a single, highly efficient enzymatic cleavage to produce any desired peptide sequence.

Method for enzymatic production of GLP-2(1-33) and GLP-2(1-34) peptides

The invention provides methods and nucleic acid constructs to express clostripain. The source of the coding region for recombinantly expressed clostripain is

Methods and constructs for high yield expression of clostripain

Method for universal enzymatic production of bioactive peptides

The invention provides a method of producing a polypeptide having a C-terminal &agr;-carboxamide group. It particularly concerns an enzymatic modification of selected substrate polypeptides which result in cleavage of the substrate polypeptide to form a product peptide with a C-terminal arginine residue having an &agr;-carboxamide group (C-terminal &ldquo;Arg-NH &rdquo;). The method includes contacting an aqueous-based solution including (i) ammonia reagent and (ii) the substrate polypeptide with (iii) clostripain.

Clostripain catalyzed amidation of peptides

A process for the recombinant preparation of a calcitonin fragment and the use of the fragment in the preparation of calcitonin and related analogs is provided. The process includes recombinantly forming a fusion protein which includes the calcitonin fragment linked to a carbonic anhydrase. The recombinantly formed fusion protein is subsequently cleaved to produce a polypeptide which includes the calcitonin fragment. A method for producing a calcitonin carba analog which includes condensing a desaminononapeptide with the recombinantly formed calcitonin fragment is also provided.

Recombinant preparation of calcitonin fragments and use thereof in the preparation of calcitonin and related analogs

The method of the invention provides for the formation of a recombinant polypeptide which has been modified at the C-terminal end through the use of a transpeptidation process. The method is suitable for modifying recombinant polypeptides of any source including those which may be commercially available, those derived from recombinant single copy or multicopy polypeptide constructs, or those derived from single or multicopy recombinant fusion protein constructs. The transpeptidation reaction involves contacting an endopeptidase enzyme with a recombinant polypeptide to substitute an addition unit, of one or more amino acids, for a leaving unit, linked to a core polypeptide through a cleavage site recognized by the endopeptidase enzyme. Recombinant polypeptides derived from multicopy polypeptide constructs may be cleaved from the multicopy polypeptide at the N-terminal and C-terminal ends and simultaneously under go substitution of the leaving unit by the desired addition unit. The invention utilizes known and newly discovered cleavage recognition sites to effectuate the desired modification products.

Enzymatic method for modification of recombinant polypeptides

A method and recombinant assay cell for detecting growth hormone releasing hormone (GHRH) in a sample, the method includes exposing a recombinant cell to the sample and measuring transcription of a reporter gene. A suitable recombinant cell includes a reporter gene operatively connected to a cAMP-responsive promoter and a GHRH-responsive protein whose binding to GHRH induces the production of cAMP. GHRH present in an assay sample results in the GHRH-responsive protein activating the production of cAMP, which then activates the c-AMP-responsive promoter to express the reporter protein. The amount of reporter protein produced is quantitatively correlated to the amount of GHRH in the sample. In one embodiment, the protein is a cell surface receptor for GHRH.

Bioassay for growth hormone releasing hormone

The invention provides customized proteases (i.e., mutant enzymes), methods of making customized proteases, as well as methods of using customized proteases. The customized proteases of the invention are derived from the known proteases. Altered transacylation reactions include the capability to perform transacylation reactions not substantially catalyzed by the known protease or the capability to perform transacylation reactions with improved yields, or both. The methods of the invention provide for customized proteases through site specific or random mutagenesis of the active site amino acids of the known proteases. The invention also provides for methods of using the customized proteases to prepare a preselected transacylation products. The preselected transacylation products produced can be modified by substitution at the N-or C-terminal with nucleophiles such as L-amino acids, D-amino acids, amino acid amides, and radioactive amino acids.

Customized proteases

The present invention is directed to a protein purification construct having three tandem, coupled segments composed of a binding protein, an interconnecting linker and a variable fused polypeptide which incorporates the one or more copies of a product peptide. The binding protein is a mammalian or human carbonic anhydrase, or a modified version of the carbonic anhydrase. The protein purification construct may be employed in methods for expression of the product peptide in microbial and higher organism and for ligand immobilized affinity purification of the product peptide.

Purification cloning of peptides

A method for the production of C-terminal amidated recombinant peptides is provided. The method employs a recombinant protein construct having multiple copies of a target peptide linked by intraconnecting peptides. The intraconnecting peptides permit the multicopy construct to be selectively reacted to produce product peptides having a C-terminal .alpha.-carboxamide. A recombinant gene containing a DNA sequence coding for the recombinant protein construct and an expression cassette, an expression vector and a transformed cell including the recombinant gene are also provided.

Production of C-terminal amidated peptides from recombinant protein

Metal binding polypeptides which include an amino acid sequence coding for a variable region of a monoclonal antibody which immunoreacts with a mercury cation and nucleotides which include a nucleic acid sequence coding for the variable region are provided. The invention is also directed to fusion proteins which include a phage coat protein or portion thereof and the monoclonal antibody heavy chain variable region. The invention also provides bacteriophages which include the fusion protein in their coat. In addition, methods for detecting, removing, adding, or neutralizing mercuric cations in biological or inanimate systems through the use of the mercury binding polypeptides are provided.

Mercury binding polypeptides and nucleotides coding therefore

Growing community of inventors

Walton, NE, United States of America

Fred W Wagner