Fibroin-Like Protein Production Method

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.

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 medium,   (B) inducing expression of the gene encoding the fibroin-like protein, and   (C) collecting the fibroin-like protein;   wherein cell proliferation after inducing the expression is reduced;   wherein the induction of the expression is performed after the cells sufficiently proliferate when OD620 of the culture medium becomes 40 to 500;   wherein said reduced cell proliferation results in cumulative specific proliferation rate of 0.070 (/h) or lower;   
       wherein the fibroin-like protein is:
 (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):
   REP1−REP2  (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 amino acids selected from 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 amino acids selected from 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 feed medium is fed during the period after inducing the expression, and wherein the ratio of a growth factor with respect to all ingredients in a feed medium fed during a period after inducing the expression is lower than 30% (w/w). 
     
     
         3 . The method according to  claim 2 , 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. 
     
     
         4 . The method according to  claim 1 , wherein the cell growth rate slope is 4.0 (%/h) or smaller, at the time point at which accumulation of the fibroin-like protein stops. 
     
     
         5 . The method according to  claim 1 , wherein the culture is performed under limitation of a growth factor during the period after inducing the expression. 
     
     
         6 . The method according to  claim 5 , wherein a feed medium is fed during the period after inducing the expression, and wherein said feed medium is not comprising said growth factor, or the amount of said growth factor with respect to all ingredients in said feed medium is less than that in the starting medium.