University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Biochemistry -- Faculty Publications Biochemistry, Department of 1993 EXOPEPTIDASE CATALYZED SITE-SPECIFIC BONDING OF SUPPORTS, LABELS AND BIOACTIVE AGENTS TO PROTEINS Fred W. Wagner Thomas R. Coolidge Sheldon M. Schuster Jay Stout Dwane E. Wylie See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/biochemfacpub Part of the Biochemistry Commons, Biotechnology Commons, and the Other Biochemistry, Biophysics, and Structural Biology Commons This Article is brought to you for free and open access by the Biochemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Biochemistry -- Faculty Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Fred W. Wagner, Thomas R. Coolidge, Sheldon M. Schuster, Jay Stout, Dwane E. Wylie, Klaus Breddam, and William Lewis US005234820A United States Patent (19) 11 Patent Number: 5,234,820 Wagner et al. (45) Date of Patent: Aug. 10, 1993 (54) EXOPEPTDASE CATALYZED (56) References Cited SITE-SPECIFICBONDING OF SUPPORTS, LABELS AND BOACTIVE AGENTS TO FOREIGN PATENT DOCUMENTS PROTENS 0085516 1/1981 European Pat. Off. (75) Inventors: Fred W. Wagner, Walton, Nebr.; OTHER PUBLICATIONS Thomas R. Coolidge, Falls Village, Kauer, et al., The Journal of Biological Chemistry, vol. Conn.; Sheldon M. Schuster, 261, No. 23, 1986, pp. 10695-10700. Gainesville, Fla.; Jay Stout; Dwane E. Wylie, both of Lincoln, Nebr.; Primary Examiner-David M. Naff Klaus Breddan, Glostrup, Denmark; Attorney, Agent, or Firm-Merchant, Gould, Smith, William Lewis, Lincoln, Nebr. Edell, Welter & Schmidt (73) Assignees: Board of Regents of the University of 57 ABSTRACT Nebraska; BioNebraska, Inc., both of An auxiliary substance such as a label, support, or bi Lincoln, Nebr. oactive agent is attached to a protein at a site that is remote from the active site of the protein by the use of 21 Appl. No.: 545,915 exopeptidase and a nucleophile which is an amino acid, amino acid derivative, amine or alcohol. In one embodi 22) Filed: Jun. 28, 1990 ment, the nucleophile is attached to the carboxy termi nus of a protein by catalysis with exopeptidase to form Related U.S. Application Data an adduct and then the adduct or its combination with (63) Continuation-in-part of Ser. No. 375,138, Jun. 30, 1989. a linker arm is bound to the auxiliary substance. In another embodiment, the auxiliary substance or its com (51) Int. Cl’...................... C12P1/00; G01N 33/532; bination with a linker arm is bound to the nucleophile to G01N 33/547; C12N 11/06 form an intermediate substance which is then coupled (52) U.S. C. ....................................... 435/41; 435/7.1; by catalysis with exopeptidase to the carboxy terminus 435/7.92; 435/181; 436/532;436/544; 530/816 of a protein. 58) Field of Search ................. 435/41, 174, 177, 181, 435/7.1, 7.92; 436/532, 544; 530/816 7 Claims, 2 Drawing Sheets U.S. Patent Aug. 10, 1993 Sheet 1 of 2 5,234,820 FG. A 12 HOURS U.S. Patent Aug. 10, 1993 Sheet 2 of 2 5,234,820 HOURS FIG 2 5,234,820 1. 2 cially applicable to immobilization, is the capability for EXOPEPTDASE CATALYZED STE SPECIFIC repeated use and for a high packing density. The fourth BONDING OF SUPPORTS, LABELS AND is the avoidance of attachment of the support, label or BOACTIVE AGENTS TO PROTENS agent within or in the vicinity of the active site of the protein. Otherwise, the resulting loss of functional ca CROSS-REFERENCE TO RELATED pacity often causes inadequate reactivity and the need APPLICATION to use more protein. This application is a continuation-in-part of U.S. pa One of the most important protein embodiments tent application Ser. No. 07/375,138, filed Jun. 30, 1989. being investigated today is the antibody. The need to 10 minimize the attachment of immobilizing supports, la TECHNICAL FIELD beling groups or bioactive agents within or near the This invention relates to immobilized and labeled antigen-binding site of an antibody is widely recog proteins and to the attachment of proteins to bioactive nized. agents. Specifically, it relates to methods for attaching One method for such minimization involves binding labels, immobilization supports and bioactive agents to 15 antigen to the antibody prior to reaction with the label specific sites of proteins. ing group, bioactive agent, or immobilizing support. In BACKGROUND OF THE INVENTION this manner, the antigen shields the antibody binding It is well-known that the function of bioactive prote site from reaction. The success of this shielding method, ins can often be enhanced by their combination with however, is limited. Although a high affinity of the other substances. When used to catalyze a reaction or to antibody for the antigen exists, the equilibrium between obtain separation, proteins can be immobilized to in the antibody/antigen complex and the free an crease reaction efficiency and simplify the processing. tibody/antigen enables free antibody to react. This has When used as detecting agents, proteins can be labeled foreseeable negative consequences. In addition, expo to facilitate measurement. When used to complex with 25 sure of the antigen to a labeling group, bioactive agent, or treat biological organisms, proteins can be combined or immobilizing support often results in attachment of with bioactive agents (hereinafter called "augmenta that material to the antigen. tion') to help achieve treatment efficacy. The known methods for immobilizing, labeling or Methods for immobilizing proteins are desirable be augmenting any kind of protein fall far short of main cause they localize reaction sites and improve economic taining the functional capacity of the protein. Protein recovery. Moreover, immobilized proteins are gener reactivity is generally lessened. Proper spatial orienta ally less susceptible to the loss of activity due to chemi tion and packing density are often lacking. And, as a cal attack and changes in temperature and pH than are result, many attendant economic, toxic, reactive and free proteins. non-specificity problems occur. Consequently, better Methods for labeling or augmenting proteins are 35 and more specific methods for binding labels, supports desirable because they facilitate quantification, localiza or bioactive agents to proteins are needed. tion, specificity and reactivity of the protein. The result It is an object of the invention, therefore, to develop ing combinations, moreover, are the resulting combina methods for labeling, immobilizing or bioactively aug tions and/or powerful tools for clinical analysis and menting proteins at sites remote from the functionally treatment. active site or sites of the protein. It is also an object of Numerous techniques exist for protein immobiliza the invention to immobilize proteins by covalently bind tion on solid supports. Proteins can be physically ad ing them to an inert immobilization support. Another sorbed onto inert supports or can be covalently bound object of the invention is to covalently bind a label to a to the support through reaction with bifunctional linker specific site of a protein. Another object is to covalently arms. Microencapsulation, gel entrapment and com 45 bind bioactive agents to a specific location on a protein. plexation (with ion exchange resins) also can bind and A further object of the invention is to immobilize prote immobilize. ins so that they have the correct spatial orientation and Numerous techniques also exist for binding labels and packing density which will allow unhindered access to bioactive agents to proteins. Most of these techniques the functionally active site. call for reaction of the label or agent and a functional group of the protein, such as an amino group, which SUMMARY OF THE INVENTION occurs repeatedly throughout the protein. Although These and other objects are achieved by the present some repetitions of such a group are shielded from invention which is directed to methods for immobiliz binding by the conformation of the protein, many others ing, labeling and augmenting proteins. Generally, the are exposed and available for binding with the labeling 55 method involves binding the immobilization support, group or bioactive agent. The result is a mixture of label or bioactive agent (hereinafter called "auxiliary proteins having labels or bioactive agents attached at substance') to a protein at a specific site so that interfer various nonspecific sites. ence with the function and performance of the protein is With any of these techniques for immobilizing, label minimized or eliminated. Preferably, this specific site is ing or augmenting, several criteria should be met. The highly remote from the active sites of the protein. first is a correct spatial orientation for optimum reactiv In particular, the method of the invention involves ity of the proteins. A protein functions best when it is two primary reactions. The first (hereinafter called the bound in a fashion that orients its active sites away from "primary coupling reaction"), catalytically couples an the support, label or bioactive agent and renders the amino acid, an amino acid derivative, an amine or an sites available for functional operation. The second is 65 alcohol (hereinafter called "nucleophile"), to the car the exhibition of protein activities and specificities that boxy terminus of the protein through the use of an are at least comparable to those exhibited