Coated substrates employing oriented layers of mutant heme proteins and methods of making and using same

• US 5,466,589 A
• Filed: 09/11/1992
• Issued: 11/14/1995
• Est. Priority Date: 11/19/1990
• Status: Expired due to Term

• Current Assignee: Board of Trustees of The University of Illinois
• Original Assignee: Biotechnology Research And Development Corporation (Dow, Inc.)
• Inventors: McLean, Mark A., Olinger, Jill M., Stayton, Patrick, Sligar, Stephen G., Bohn, Paul W.
• Primary Examiner(s): Walsh, Stephen G.
• Application Number:
  US07/945,291
• Time in Patent Office: 1,159 Days
• Field of Search: 435/174, 435/189, 435/288, 435/817, 435/808, 435/969, 436/164, 436/518, 436/801, 436/805, 436/806, 422/57, 422/70, 422/76, 422/82.01, 422/82.05, 530/385, 530/810, 530/401
• US Class Current: 435/174
• CPC Class Codes: C07K 14/80 Cytochromes
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10S 530/81 Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier

Abstract

The present invention is directed to coated substrates having a coating of biological macromolecules, preferably proteins, which are capable of being immobilized on a substrate surface and have a marker. These proteins usually are mutant proteins obtained by mutagenesis of the gene encoding a random positioning protein. When a mutant protein molecule is immobilized on the substrate, the marker of the mutant protein molecule is in a select spatial relationship with both the substrate and the markers of adjacent protein molecules. A substrate coated with an oriented layer of the mutant proteins exhibits improved or different properties when compared to a substrate having a randomly positioned layer of proteins thereon.

Claims

1. A coated substrate comprising

a substrate having at least a first portion having a surface;

and

a plurality of mutant heme protein molecules having heme prosthetic groups and preferential binding sites, wherein said preferential binding site of each mutant heme protein molecule was provided by replacement of a surface amino acid residue and said preferential binding site is a reacted amino acid residue coupled with a functional group of said surface of said first portion, such that a coating of said mutant heme protein molecules is formed on said surface of said first portion, and the heme prosthetic groups of said coating of said surface of said first portion have the same spatial relationship with respect to to said surface of said first portion.

2. A coated substrate according to claim 1, wherein said substrate further comprises a second portion having a surface.

3. A coated substrate according to claim 1, wherein said mutant heme proteins are mutated forms of heme proteins selected from the group consisting of cytochrome a, cytochrome b, cytochrome c, myoglobin, hemoglobin or cytochrome P-450.

4. A coated substrate according to claim 1, wherein said functional group is provided by a derivatization reagent.

5. A coated substrate according to claim 1, wherein said substrate comprises a portion having a second mutant protein immobilized thereon.

6. A sensor comprising a coated substrate according to claim 1.

7. A chromatography system comprising a coated substrate according to claim 1.

8. A conductive device comprising a coated substrate according to claim 1.

9. A phase grating comprising a coated substrate according to claim 1.

10. A light modulator comprising a coated substrate according to claim 1.

11. A coated substrate according to claim 1, wherein said mutant heme protein molecules have a site for binding ligands, reporter groups or proteins.

12. A coated substrate according to claim 11, wherein said mutant heme protein molecules having said site for binding are immobilized on said surface to form a first monomolecular layer on said portion, and mutant heme protein molecules are bound to said site for binding to form a second monomolecular layer on said coated substrate.

13. A coated substrate according to claim 2, wherein said surface of said second portion comprises a plurality of mutant heme protein molecules having heme prosthetic groups and preferential binding sites, wherein said preferential binding site of each mutant heme protein molecule has a reacted amino acid residue coupled with a functional group of said surface of said second portion, such that a coating of said mutant heme protein molecules is formed on said surface of said second portion, and the heme prosthetic groups of said coating on said surface of said second portion have the same spatial relationship with respect to said surface of said second portion.

14. A coated substrate according to claim 13, wherein said spatial relationship of said first portion is different than said spatial relations of said second portion.

15. A coated substrate according to claim 1, wherein said heme prosthetic groups of said coating have the same spatial relationship with respect to adjacent heme prosthetic groups of said coating.

16. A coated substrate comprising

a substrate having at least one portion having a surface, and

a plurality of mutant heme protein molecules immobilized on said surface to form a coating, wherein (A) said mutant heme protein molecules have heme prosthetic groups and preferential binding sites, wherein said preferential binding site of each mutant heme protein molecule was provided by replacement of a surface amino acid residue and said preferential binding site is a reacted amino acid residue coupled with a functional group of said surface to immobilize said mutant heme protein molecule, and (B) said heme prosthetic groups of immobilized mutant heme protein molecules have select spatial relationships with respect to said surface and with respect to adjacent heme prosthetic groups.

17. A process for making a substrate having a coating of mutant heme protein molecules, comprising the steps of:

mutating a heme protein at a select position by replacement of a surface amino acid residue to obtain a preferential binding site on said mutant heme protein; and

immobilizing a plurality of said mutant heme proteins on said substrate by reaction of said preferential binding site with a functional group on said substrate so that heme prosthetic groups of said mutant heme proteins have the same spatial relationship with respect to said surface.