Bionebraska, Inc.

University of Nebraska

Dwane E Wylie

Fred W Wagner

Sheldon M Schuster

Jay S Stout

Thomas R Coolidge

William Lewis

Gino Van Heeke

Osvaldo Lopez

Randall R Carlson

Malcolm Riddell

Klaus Breddam

Peter Goebel

Peter Joseph Murray

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

Metal binding polypeptides which include an amino acid sequence coding for a light chain variable region of a monoclonal antibody capable of immunoreacting with a lead cation and nucleotides which include a nucleic acid sequence coding for the variable region are provided. The invention is also directed to fusion proteins and Fab fragments which include the light chain variable region.

Lead binding polypeptides and nucleotides coding therefore

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

The invention is directed to monoclonal antibodies, their fragments, single chains and polypeptide mimics of their hypervariable regions which immunoreact with bare small moieties such as metallic cations and small organic molecules, the hybridomas for production of the monoclonal antibodies, immunogen compounds for developing the hybridomas, and methods for use of the monoclonal antibodies.

Metallic cation binding polypeptides and methods therefor

A method is provided for preparing a labeled protein, immobilized protein or protein-bioactive agent composition by attaching a label, support or bioactive agent to a protein by exopeptidase catalysis at a site that is remote from the active site of the protein. More specifically, an amine or alcohol group of an amino acid, amine or alcohol nucleophile is reacted by exopeptidase catalysis with a C-terminus carboxylic acid group of a protein such as an antibody, enzyme or hormone to couple the nucleophile to the protein to form an adduct, and the adduct is bound to an auxiliary substance such as a support, label or bioactive agent or its combination with a linker arm by reacting a reactive substituent of the nucleophile with a reactive group of the auxiliary substance. Alternatively, the nucleophile is bound to the auxiliary substance or its combination with a linker arm to form an intermediate, and the intermediate is coupled by exopeptidase catalysis to the protein. The exopeptidase may be a serine or cysteine exocarboxypeptidase.

Exopeptidase catalyzed site-specific bonding of supports, labels and

The invention is directed to monoclonal antibodies, their fragments, single chains and polypeptide mimics of their hypervariable regions which immunoreact with bare small moieties such as metallic cations the hybridomas for production of the monoclonal antibodies, immunogen compounds for developing the hybridomas, and methods for use of the monoclonal antibodies.

Monoclonal antibodies specific for metallic cations and method therefor

The invention provides method and kits for detecting a metallic cation in a sample of a body fluid. The preferred method and kits include the use of at least two different types of antibodies having different specificities. In the preferred method, the sample of body fluid can be contacted with an effective amount of a capture antibody specific for a naturally occurring polypeptide that can bind the metallic cation to form a first antigen-antibody complex. An effective amount of an antibody specific for an epitope on a metallic cation-naturally occurring polypeptide complex or an antibody specific for a metallic cation is added to the first antigen-antibody complex to form a second antigen-antibody complex. The amount of the metallic cation in the sample of body fluid is determined by detecting the amount of the second antigen-antibody complex.

Monoclonal antibody assay and kit for detecting metal cations in body

Methods are presented for producing and purifying a variable fusion polypeptide which can be purified by affinity chromatography with the binding protein partner. The variable fusion polypeptide construct has tandem coupled segments containing one or more copies of a desired peptide linked to carbonic anhydrase as the purification binding protein. In the methods, the fusion protein is expressed in a recombinant host using a recombinant vector containing a gene encoding the fusion polypeptide. Then the expressed fusion polypeptide is purified by immobilized reversible inhibitor affinity chromatography. Finally, the purified fusion polypeptide is cleaved from the desired peptides by chemical or enzymatic means and the desired peptides purified with affinity chromatography.

Purification directed cloning of peptides using carbonic anhydrase as

The invention is directed to monoclonal antibodies which immunoreact with bare small moieties such as metallic cations and small organic molecules, the hybridomas for production of the monoclonal antibodies, immunogen compounds for developing the hybridomas, and methods for use of the monoclonal antibodies.

Monoclonal antibodies specific for small moieties and method for their

The invention is directed to a method of purifying sequentially synthesized peptides and oligonucleotides by affinity techniques. Selected products are capped with and N-terminus capping agent for peptides or a 5'-terminus capping agents for oligonucleotides, and then bound with affinity agents that are selective for the corresponding capping agents.

Sequential oligonucleotide syntheses using immunoaffinity techniques

The invention provides a means for attaching a label, support or bioactive agent to a protein with an exopeptidase at a site that is remote from the active site of the protein. More specifically the invention is directed to a method for the attachment of an amino acid, amine and alcohol nucleophile to the carboxyl terminus of a protein. In one embodiment, a labeled nucleophile is attached to a protein such as an antibody. In other embodiments, the invention is directed to a method for the attachment of a protein to an immobilization support and to a method for the attachment of a bioactive agent to a protein.

An auxiliary substance such as a label, support, or bioactive agent is attached to a protein at a site that is remote from the active site of the protein by the use of exopeptidase and a nucleophile which is an amino acid, amino acid derivative, amine or alcohol. In one embodiment, the nucleophile is attached to the carboxy terminus of a protein by catalysis with exopeptidase to form an adduct and then the adduct or its combination with a linker arm is bound to the auxiliary substance. In another embodiment, the auxiliary substance or its combination with a linker arm is bound to the nucleophile to form an intermediate substance which is then coupled by catalysis with exopeptidase to the carboxy terminus of a protein.

Sequential peptide and oligonucleotide syntheses using immunoaffinity

The invention is directed to a method for purifying sequentially synthesized peptides and oligonucleotides by immunoaffinity techniques. Selected products are lapped with an antigenic capping agent and are conjugated with antibodies that are specific for the capping agent.

Growing community of inventors

Lincoln, NE, United States of America

Dwane E Wylie