US 6,958,223 B2 · Methods for producing extracellular matrix proteins

Abstract

Incorporation of certain amino acid analogs into polypeptides produced by cells which do not ordinarily provide polypeptides containing such amino acid analogs is accomplished by subjecting the cells to growth media containing such amino acid analogs. The degree of incorporation can be regulated by adjusting the concentration of amino acid analogs in the media and/or by adjusting osmolality of the media. Such incorporation allows the chemical and physical characteristics of polypeptides to be altered and studied. In addition, nucleic acid and corresponding proteins including a domain from a physiologically active peptide and a domain from an extracellular matrix protein which is capable of providing a self-aggregate are provided. Human extracellular matrix proteins capable of providing a self-aggregate collagen are provided which are produced by prokaryotic cells. Preferred codon usage is employed to produce extracellular matrix proteins in prokaryotics.

Claims18

Dependent claims

A method of producing an Extracellular Matrix Protein (EMP) or fragment thereof capable of providing a self aggregate in a cell which does not ordinarily hydroxylate proline comprising:

providing a nucleic acid sequence encoding the EMP or fragment thereof which has been optimized for expression in the cell by substitution of codons preferred by the cell for naturally occurring codons not preferred by the cell;
incorporating the nucleic acid sequence into the cell;
providing hypertonic growth media containing at least one amino acid selected from the group consisting of trans-4-hydroxyproline and 3-hydroxyproline;
and
contacting the cell with the growth media wherein the at least one amino acid is assimilated into the cell and incorporated into the EMP or fragment thereof,
wherein the EMP or fragment thereof having the at least one amino acid self aggregates to form an extracellular matrix.

3 more elements

Dependencies are inferred from the claim text.

Methods for producing extracellular matrix proteins

Life of this patent

Methods for producing extracellular matrix proteins

Life of this patent

Abstract

Claims18