Biosynthetically generated pyrroline-carboxy-lysine and site specific protein modifications via chemical derivatization of pyrroline-carboxy-lysine and pyrrolysine residues

Patent metadata

Field	Value
Publication number	US-9624520-B2
Application number	US-201414279085-A
Country	US
Kind code	B2
Filing date	May 15, 2014
Priority date	Oct 24, 2008
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

Disclosed herein is pyrroline-carboxy-lysine (PCL), a pyrrolysine analog, which is a natural, biosynthetically generated amino acid, and methods for biosynthetically generating PCL. Also disclosed herein are proteins, polypeptides and peptides that have PCL incorporated therein and methods for incorporating PCL into such proteins, polypeptides and peptides. Also disclosed herein is the site-specific derivatization of proteins, polypeptides and peptides having PCL or pyrrolysine incorporated therein. Also disclosed herein is the crosslinking of proteins, polypeptides and peptides having PCL or pyrrolysine incorporated therein.

First claim

We claim:

1. A process for preparing a protein having the structure of Formula (I):
R 1 -(AA) n -R 2
wherein:
R 1 is H or an amino terminus modification group;
R 2 is OH or a carboxy terminus modification group;
n is an integer from 1 to 5000;
each AA is independently selected from an amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (A-2) and a pyrrolysine analogue amino acid residue having the structure of Formula (B-2);

and
wherein at least one AA is a pyrrolysine analogue amino acid residue having the structure of Formula (A-2) or Formula (B-2);

the process comprising:
biosynthetically generate within a cell an amino acid of Formula (VII) or Formula (VIII):

wherein the cell comprises a pylC gene, and a pylD gene, and the cell is in contact with a growth medium comprising a precursor,
or
wherein the cell comprises a pylB gene, a pylC gene and a pylD gene, and
the cell is in contact with a growth medium comprising a precursor, incorporating the amino acid of Formula (VII) or Formula (VIII) into the protein within the cell using an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O—RS), wherein the O—RS aminoacylates the O-tRNA with the amino acid of Formula (VII) or Formula (VIII) and the O-tRNA recognize at least one selector codon of a mRNA in the cell,

and
recovering the protein.

2. The process of claim 1 , wherein the cell further comprises a pylS gene and a pylT gene and the amino acid of Formula (VII) or Formula (VIII) is incorporated into the protein within the cell by an aminoacyl tRNA synthetase and a tRNA which recognizes at least one selector codon of a mRNA in the cell, wherein the aminoacyl tRNA synthetase is a gene product of the pylS gene and the tRNA is a gene product of the pylT gene.

3. The process of claim 2 , wherein the selector codon is a TAG amber codon.

4. The process of claim 1 , wherein the precursor is D-ornithine, D-arginine, (2S)-2-amino-6-(2,5-diaminopentanamido)hexanoic acid or (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid.

5. The process of claim 1 , wherein the cell is a prokaryotic cell or a eukaryotic cell.

6. The process of claim 5 , wherein the cell is an Escherichia coli cell, a mammalian cell, a yeast cell or an insect cell.

7. The process of claim 6 , wherein the mammalian cell is a CHO cell, a HeLa cell or a HEK293F cell and the insect cell is a sf9 cell.

Assignees

• Geierstanger Bernhard
• Ou Weijia
• Cellitti Susan E
• Uno Tetsuo
• Crossgrove Tiffany
• Chiu Hsien-Po
• Grunewald Jan
• Hao Xueshi
• Novartis Ag

Inventors

• Geierstanger Bernhard
• Ou Weijia
• Cellitti Susan E
• Uno Tetsuo
• Crossgrove Tiffany
• Chiu Hsien-Po
• Grunewald Jan
• Hao Xueshi

Classifications

• C07D207/20

  with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms · CPC title

• C12P21/00 Primary

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C07D207/48

  Sulfur atoms · CPC title

• C07K5/0215

  containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu · CPC title

• C07K2/00 Primary

  Peptides of undefined number of amino acids; Derivatives thereof · CPC title