Overexpression of aminoacyl-tRNA synthetases for efficient production of engineered proteins containing amino acid analogues

• US 6,586,207 B2
• Filed: 01/23/2001
• Issued: 07/01/2003
• Est. Priority Date: 05/26/2000
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Kiick, Kristi Lynn, Tirrell, David A.
• Primary Examiner(s): Guzo, David
• Application Number:
  US09/767,515
• Publication Number:
  US 20020042097A1
• Time in Patent Office: 889 Days
• Field of Search: 435/320.1, 435/69.1, 435/325, 435/6, 435/183, 435/348, 435/366, 435/410, 435/419, 435/254.1, 435/254.11, 435/243, 435/252.3, 530/350
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 9/003 Dihydrofolate reductase [DHFR] (1.5.1.3)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

Methods for producing modified polypeptides containing amino acid analogues are disclosed. The invention further provides purified dihydrofolate reductase polypeptides, produced by the methods of the invention, in which the methionine residues have been replaced with homoallyglycine, homoproparglycine, norvaline, norleucine, cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid, 2-butynylglycine and allylglycine.

Claims

1. A method for producing a modified polypeptide, the polypeptide being modified by replacing a selected amino acid with a desired amino acid analogue, which method comprises:

a) transforming a host cell with;

i) a vector having a polynucleotide sequence encoding an aminoacyl-tRNA synthetase for the selected amino acid; and

ii) a vector having a polynucleotide sequence encoding a polypeptide molecule or interest so as to produce a host vector system;

wherein the vectors of (i) and (ii) may be the same or different

b) growing the host-vector system in a medium which comprises the selected amino acid so that the host vector system overexpresses the aminoacyl-tRNA synthetase;

c) replacing the medium with a medium which lacks the selected amino acid and has the desired amino acid analogue;

d) growing the host vector system in the medium which lacks the selected amino acid and has the desired amino acid analogue under conditions so that the host vector system overexpresses the polypeptide molecule of interest and the selected amino acid is replaced with the desired amino acid analogue thereby producing the modified polypeptide.

2. The method of claim 1 wherein the overexpression of an aminoacyl-tRNA synthetase results in an increase in the activity of the aminoacyl-tRNA synthetase.

3. The method of claim 1 wherein said host cell is from an organism selected from the group consisting of bacterial, yeast, mammalian, insect, and plant.

4. The method of claim 1 wherein said host cell is an auxotroph, wherein the auxotrophic host cell is incapable of producing the selected amino acid.

5. The method of claim 1, wherein said aminoacyl-tRNA synthetase is naturally occurring or genetically engineered.

6. The method of claim 1, wherein said aminoacyl-tRNA synthetase is methionyl tRNA synthetase.

7. The method of claim 1, wherein said selected amino acid is methionine.

8. The method of claim 1, wherein said desired amino acid analogue comprises side chain functionalities different from its corresponding natural amino acid.

9. The method of claim 1, wherein the polypeptide is dihydrofolate reductase protein.

10. The method of claim 1, wherein the selected amino acid is methionine;

and the desired amino acid analogue is selected from the group consisting of homoallyglycine, homoproparglycine, norvaline, norleucine, cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid, 2-butynylglycine, allylglycine, azidoalanine and azidohomoalanine.

11. The method of claim 4 wherein said auxotrophic host cell is from an organism selected from the group consisting of bacterial, yeast, mammalian, insect, and plant.

12. The method of claim 11 wherein said auxotrophic host cell is a methionine auxotroph.

13. The method of claim 8, wherein said amino acid analogue is a hydrophobic amino acid analogue.

14. The method of claim 13, wherein the hydrophobic amino acid analogue is selected from the group consisting of fluorinated, electroactive, and unsaturated amino acids.