Surface display of selenocysteine-containing peptides

• US 7,141,366 B1
• Filed: 05/12/2000
• Issued: 11/28/2006
• Est. Priority Date: 05/14/1999
• Status: Expired due to Fees

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Sandman, Karen E., Noren, Christopher J.
• Primary Examiner(s): Paras, Jr., Peter
• Assistant Examiner(s): Lundgren, Jeffrey S
• Application Number:
  US09/937,187
• Time in Patent Office: 2,391 Days
• Field of Search: 435/5, 435/7.1, 435/7.4, 435/69.1, 435/69.2, 435/70.1, 435/963, 530/350
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

The naturally-occurring amino acid selenocysteine (SEC) is incorporated uniquely and specifically in the context of a polypeptide displayed on the surface of an amplifiable genetic particle (phage, cell or spore) in response to incorporation signals engineered in the encoding DNA. In addition to conferring the unique activities of the selenol group to the chemistry of the displayed peptide, Sec also provides a unique handle for specific chemical modification of the display peptide. In addition to increasing the palette of available residues in a random peptide library to 21 possibilities, present invention also provides a means of tethering virtually any desired chemical functionality to the incorporated Sec.

Claims

1. A fusion protein, comprising:

a selenocysteine-containing peptide fused to a surface protein displayed on an amplifiable genetic particle selected from a virus and a cell.

2. A fusion protein according to claim 1, wherein the covalent linkage between the selenocysteine-containing peptide and the surface protein is a peptide bond.

3. A fusion protein according to claim 1, wherein the selenocysteine-containing peptide is expressed by a DNA having a TGA codon and a selenocysteine insertion sequence.

4. A fusion protein according to claim 3, wherein the selenocysteine insertion sequence is located downstream of the TGA codon.

5. A fusion protein according to claim 1, wherein the selenocysteine is flanked on either or both sides by one or more randomized amino acids.

6. A fusion protein according to claim 1, wherein the selenocysteine in the peptide is positioned adjacent to one side of one or more randomized amino acids, the one or more randomized amino acids being flanked on a second side by a cysteine.

7. A fusion protein according to claim 1, wherein the selenocysteine in the peptide is capable of chemical derivatization of the selenol group.

8. A fusion protein according to claim 7, wherein the chemical derivatization results from a nucleophilic substitution reaction.

9. A fusion protein according to claim 7, wherein the chemical derivatization results from an oxidation reaction.

10. A fusion protein according to claim 7, wherein the chemical derivatization results from a metal coordination reaction.

11. A fusion protein according to claim 7, wherein a product of chemical derivatization of the selenocysteine in the peptide is a chemical functionality selected from the group consisting of enzyme substrates, enzyme cofactors, enzyme inhibitors, receptor ligands and cytotoxic agents.

12. A fusion protein according to claim 1, wherein the selenocysteine-containing peptide further comprises an enzyme substrate or is modified at the selenocysteine to form an enzyme substrate.

13. A fusion protein according to claim 12, wherein the enzyme substrate forms a reaction product in the presence of an enzyme and the enzyme substrate is located on the surface of the amplifiable genetic particle.

14. A fusion protein of claim 13, wherein the reaction product is capable of binding to an affinity substrate.

15. A fusion protein, according to claim 13, wherein the recombinant protein is selected from a library of variants of a single enzyme, wherein each variant contains one or more amino acid substitutions relative to the single enzyme.

16. A fusion protein according to claim 13, wherein the recombinant protein is selected from an expressed c-DNA library.