Fibroin-like protein production method

Patent metadata

Field	Value
Publication number	US-10752660-B2
Application number	US-201615356915-A
Country	US
Kind code	B2
Filing date	Nov 21, 2016
Priority date	May 21, 2014
Publication date	Aug 25, 2020
Grant date	Aug 25, 2020

Abstract

A method for producing a fibroin-like protein is described. A fibroin-like protein is produced by culturing Escherichia coli having a gene encoding the fibroin-like protein in a medium, inducing expression of the gene encoding the fibroin-like protein, and collecting the fibroin-like protein, wherein the cell proliferation after inducing the expression is reduced.

First claim

The invention claimed is:

1. A method for producing a fibroin-like protein, the method comprising:
(A) culturing an Escherichia coli bacterium having a gene encoding the fibroin-like protein in a culture medium;
(B) inducing expression of the gene encoding the fibroin-like protein;
(C) after said inducing, a feed medium is added to said culture medium, wherein said feed medium comprises a reduced amount of or no growth factor, resulting in reduced cell proliferation; and
(D) collecting the fibroin-like protein;
wherein said reduced amount of the growth factor with respect to all ingredients in said feed medium is less than 30% (w/w),
wherein in step (C), said reduced cell proliferation results in cumulative specific proliferation rate of 0.070 (/h) or lower,
wherein the fibroin-like protein comprises:
(a) a fibroin; or
(b) a fibrous protein having a sequence similar to a repetitive sequence of a fibroin,
wherein the sequence similar to a repetitive sequence of a fibroin is a sequence represented by the following formula (I),
REP 1− REP 2  (I)

wherein the protein (b) has the sequence represented by the formula (I) at the repetition number of 2 or more,
wherein, independently for each repetition, the REP1 is an amino acid sequence consisting of a continuous sequence of one or more of the amino acids alanine and glycine, and the length of the REP1 is 2 to 20 residues, and
wherein, independently for each repetition, the REP2 is an amino acid sequence comprising one or more of the amino acids glycine, serine, glutamine, and alanine, the total number of glycine, serine, glutamine, and alanine residues is 40% or more of the total number of amino acid residues of the REP2, and the length of the REP2 is 2 to 200 residues.

2. The method according to claim 1 , wherein a parameter selected from the group consisting of a) the cell growth rate, b) cell growth rate slope, c) cumulative specific proliferation rate, and d) combinations thereof, is/are reduced after inducing the expression.

3. The method according to claim 1 , wherein the growth factor is selected from the group consisting of a) a carbon source, b) nitrogen source, c) phosphate source, d) sulfur source, e) mineral, f) nutrient required because of auxotrophy, and g) combinations thereof.

4. The method according to claim 1 , wherein the growth factor is selected from the group consisting of a) a carbon source, b) organic nitrogen source, c) phosphate source, and d) combinations thereof.

5. The method according to claim 1 , wherein a ratio of a carbon source with respect to all ingredients in the feed medium fed during a period after inducing the expression is 70% (w/w) or higher.

6. The method according to claim 1 , wherein said culturing is performed so that the cumulative specific carbon source consumption rate is 0.35 g/g/hr or lower after inducing the expression.

7. The method according to claim 1 , wherein the cell proliferation after inducing the expression is reduced by allowing the cells to sufficiently proliferate before inducing the expression.

Assignees

• Ajinomoto Kk
• Spiber Inc

Inventors

• Nakase Kentaro

Classifications

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N1/00

  Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title

• C07K14/43518 Primary

  from spiders · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title