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Last Updated: March 28, 2024

Claims for Patent: 5,935,823


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Summary for Patent: 5,935,823
Title: Totally synthetic affinity reagents
Abstract:A novel process for producing novel and/or improved heterofunctional binding fusion proteins termed Totally Synthetic Affinity Reagents (TSARS) is disclosed. TSARs are concatenated heterofunctional polypeptides or proteins comprising at least two functional regions: a binding domain with affinity for a ligand and a second effector peptide portion that is chemically or biologically active. In one embodiment, the heterofunctional polypeptides or proteins further comprise a linker peptide portion between the binding domain and the second active peptide portion. The linker peptide can be either susceptible or not susceptible to cleavage by enzymatic or chemical means. Novel and/or improved heterofunctional binding reagents as well as methods for using the reagents for a variety of in vitro and in vivo applications are also disclosed.
Inventor(s): Fowlkes; Dana M. (Chapel Hill, NC), Kay; Brian K. (Chapel Hill, NC)
Assignee: The University of North Carolina at Chapel Hill (Chapel Hill, NC)
Application Number:08/420,945
Patent Claims:1. A method for identifying a heterofunctional fusion protein having specificity for a ligand of choice, comprising:

(a) inserting into a plurality of vectors (i) one or more of a plurality of different first nucleotide sequences, each first nucleotide sequence comprising a sequence encoding a putative binding domain, said sequence encoding the putative binding domain having been generated totally de novo by random chemical synthesis, and (ii) a second nucleotide sequence encoding a biologically or chemically active effector domain, in which each first nucleotide sequence/second nucleotide sequence combination is located downstream from a 5' ATG start codon to produce a library of vectors coding for in-frame fusion proteins;

(b) transforming compatible host cells with the vectors formed in step (a) to express the fusion proteins; and

(c) screening the expressed fusion proteins to identify a fusion protein having binding specificity for the ligand of choice and the desired biological or chemical activity of said effector domain, in which the ligand is selected from the group consisting of a chemical group, an ion, a metal, a peptide or any portion thereof, a nucleic acid or any portion thereof, a carbohydrate, carbohydrate polymer or portion thereof, a lipid, a fatty acid, a viral particle or portion thereof, a membrane vesicle or portion thereof, a cell wall component, a synthetic organic compound, and an inorganic compound.

2. The method according to claim 1, in which the the fusion protein having specificity for a ligand choice is detected by means of the biological or chemical activity of the effector domain encoded by the second nucleotide sequence.

3. The method according to claim 1, in which step (a) further comprises inserting a third nucleotide sequence encoding a linker domain between the first and second nucleotide sequences.

4. The method according to claim 3, in which the linker domain is stable.

5. The method according to claim 3, in which the linker domain moiety is susceptible to cleavage by enzymatic or chemical means.

6. The method according to claim 1, in which the biologically or chemically active effector domain is selected from the group consisting of detectable, enzymatic and therapeutically active polypeptide or protein moieties.

7. The method according to claim 6, in which the biologically or chemically active effector domain is .beta.-galactosidase or a portion thereof.

8. The method according to claim 5, in which the linker domain is susceptible to cleavage by enzymatic means.

9. The method according to claim 8, in which the enzymatic means is selected from the group consisting of collagenase, enterokinase, Factor Xa and thrombin.

10. The method according to claim 5, in which the linker peptide moiety is susceptible to cleavage by chemical means.

11. The method according to claim 10, in which the chemical means is cyanogen bromide.

12. The method according to claim 1, in which the vector is selected from the group consisting of bacterial plasmid, bacterial phage, eukaryotic plasmid and eukaryotic viral vectors.

13. The method according to claim 12, in which the vector is selected from the group consisting of p340, pBR322, pAC1005, pSC101, pBR325, lambda, M13, T7, T4, SV40, EBV, adenovirus, vaccinia, yeast vectors, insect vectors, and derivatives thereof.

14. The method according to claim 13, in which the vector is p340.

15. A method for producing a unifunctional polypeptide or protein having specificity for a ligand of choice, comprising: chemically synthesizing the polypeptide or protein having an amino acid sequence of the binding domain of a fusion protein identified according to the method of claim 1.

16. A method for producing a unifunctional polypeptide or protein having specificity for a ligand of choice, comprising recovering the heterofunctional fusion protein identified according to claim 5, and cleaving said unifunctional polypeptide or protein by enzymatic or chemical means.

17. A method for identifying a heterofunctional fusion protein having specificity for a ligand of choice, comprising:

screening fusion proteins expressed by host cells transformed with a library of vectors coding for in-frame fusion proteins to express the fusion proteins, said library being formed by inserting into a plurality of vectors (i) one or more of a plurality of different first nucleotide sequences, each first nucleotide sequence comprising a sequence encoding a putative binding domain, said sequence encoding the putative binding domain having been generated totally de novo by random chemical synthesis, and (ii) a second nucleotide sequence encoding a biologically or chemically active effector domain, in which each first nucleotide sequence/second nucleotide sequence combination is located downstream from a 5' ATG start codon to produce said library of vectors coding for in-frame fusion proteins to identify a fusion protein having binding specificity for the ligand of choice and the desired biological or chemical activity, in which the ligand is selected from the group consisting of a chemical group, an ion, a metal, a peptide or any portion thereof, a nucleic acid or any portion thereof, a carbohydrate, carbohydrate polymer or portion thereof, a lipid, a fatty acid, a viral particle or portion thereof, a membrane vesicle or portion thereof, a cell wall component, a synthetic organic compound, and an inorganic compound.

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