| CPC C07K 14/605 (2013.01) [C12N 15/70 (2013.01); C12P 21/005 (2013.01); C07K 2319/02 (2013.01)] | 3 Claims |
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1. A method for continuous production and secretion of recombinant GLP-1 peptide by E. coli, comprising the steps of:
a) transforming E. coli with an expression vector encoding recombinant GLP-1 peptide to produce recombinant E. coli;
b) preparing a starter culture of recombinant E. coli by growing the culture at 37° C. with 225 rpm for 12 hours in a starter culture growth media until the OD600 of the starter culture reaches 5.0-6.0;
c) preparing a perfusion-based fermenter system by adding initial batch media to the fermenter vessel comprising of glucose/dextrose at a concentration of 10 g/L and maintaining the pH at 6.9;
d) adding the starter culture to the fermenter vessel and maintaining the pH at 6.9;
e) adding lac operon inducing to the fermenter vessel when the residual glucose/dextrose concentration in the initial batch media has reduced to ˜5 g/L for the induction of production and secretion of recombinant GLP-1 peptide from recombinant E. coli; and
f) initiating perfusion-based fermentation system after 30-40 mins of induction for separating the recombinant E. coli as retentate from the spent culture media containing the secreted recombinant GLP-1 peptide as permeate, harvesting recombinant GLP-1 peptide from the permeate, and re-feeding the fermenter vessel with fresh perfusion media and with the retentate recombinant E. coli for continuous production and secretion of recombinant GLP-1 peptide;
wherein, the expression vector consists of SEQ ID NO: 6, wherein, said expression vector includes: the DNA sequence consisting of SEQ ID NO: 1 which encodes the recombinant GLP-1 peptide, the DNA sequence consisting of SEQ ID NO: 3 which encodes the secretory signal peptide of the gene ompf, and a DNA sequence encoding a truncated YebF peptide of SEQ ID NO:5;
wherein, the expression vector, encoding recombinant GLP-1 peptide, of SEQ ID NO: 6 secretes recombinant GLP-1 peptide in the range of 1-1.2 g/L/hr by a perfusion-based fermentation system;
wherein, the initial batch media is a chemically defined media comprising of: 139 mM glucose/dextrose; 30.2 mM diammonium phosphate; 54.29 mM glycerol as stabilizing agent; 8.84 mM citric acid; 1 mM glycine as essential amino acid; 1 mM arginine as positively charged amino acid; 0.06 mM thiamine; 5 mM magnesium sulfate heptahydrate; 97.73 mM potassium dihydrogenphosphate; 2.13 mM sodium chloride; 0.09 mM calcium chloride; and salts of trace elements Fe (III) citrate at 1-5g/L, CoCl2-6H2O at 0.1-2 g/L, MnCl2-4H2O at 0.5-5 g/L, CuCl2-2H2O 0.01-1 g/L, H3BO3 at 0.1-1 g/L, Na2MoO4-2H2O at 0.01-1 g/L, Zn acetate-2H2O at 0.5-5 g/L concentrations; and chelating agent EDTA at 0.01-5 g/L concentration;
and
wherein, the perfusion media is a chemically defined media comprising of 13.9 mM glucose/dextrose; 30.2 mM diammonium phosphate; 54.29 mM glycerol as stabilizing agent; 8.84 mM citric acid; 1 mM glycine as essential amino acid; 1 mM arginine as positively charged amino acid; 0.06 mM thiamine; 5 mM magnesium sulfate heptahydrate; 97.73 mM potassium dihydrogenphosphate; 2.13 mM sodium chloride; 0.09 mM calcium chloride; and
salts of trace elements Fe (III) citrate at 1-5 g/L, CoCl2-6H2O at 0.1-2 g/L, MnCl2-4H2O at 0.5-5 g/L, CuCl2-2H2O 0.01-1 g/L, H3BO3 at 0.1-1 g/L, Na2MoO4-2H2O at 0.01-1 g/L, Zn acetate-2H2O at 0.5-5 g/L concentrations; and chelating agent EDTA at 0.01-5 g/L concentration.
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