Method for continuous production of recombinant GLP-1 peptide by bacteria
Inventors
LOKIREDDY, Sudarsanareddy • KONA, VENKATA SRI KRISHNA
Assignees
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Publication Number
US-12152062-B2
Publication Date
2024-11-26
Expiration Date
Abstract
The invention relates to a method for continuously producing and secreting recombinant Glucagon-like peptide-1 (GLP-1) by bacteria, more specifically E. coli. More specifically, the invention relates to use of novel bacterial expression vector for producing and enabling extracellular secretion of GLP-1, use of novel media composition for enhancing the secretion and enabling purification, and a perfusion-based fermentation system for continuous production and separation of recombinant GLP-1 peptide.
Core Innovation
The invention relates to continuous production and secretion of recombinant GLP-1 peptide by E. coli using a perfusion-based fermentation system. Recombinant E. coli are produced by transforming E. coli with an expression vector consisting of SEQ ID NO: 6, including SEQ ID NO: 1 encoding the recombinant GLP-1 peptide, SEQ ID NO: 3 encoding the secretory signal peptide of the gene ompf, and a DNA sequence encoding a truncated YebF peptide of SEQ ID NO: 5.
A perfusion-based fermenter system is prepared with initial batch media and chemically defined perfusion media, and pH is maintained at 6.9. Induction is carried out by adding lac operon inducing when residual glucose/dextrose is reduced to approximately 5 g/L for induction of production and secretion of recombinant GLP-1 peptide from recombinant E. coli.
After induction, perfusion-based fermentation is initiated to separate recombinant E. coli as retentate from spent culture media containing the secreted recombinant GLP-1 peptide as permeate, while harvesting recombinant GLP-1 peptide from the permeate. The separated retentate recombinant E. coli is re-fed into the fermenter vessel with fresh perfusion media for continuous production and secretion of recombinant GLP-1 peptide.
Claims Coverage
The independent claim describes a continuous production and secretion method for recombinant GLP-1 peptide by recombinant E. coli using a perfusion-based fermentation system. The inventive features include the specific expression vector design, induction tied to residual glucose/dextrose, and continuous retentate/permeate separation with chemically defined media and a pH setpoint.
Continuous GLP-1 secretion in perfusion-based fermentation with retentate/permeate separation
Perfusion-based fermentation separates recombinant E. coli as retentate from spent culture media containing secreted recombinant GLP-1 peptide as permeate, harvests recombinant GLP-1 peptide from the permeate, and re-feeds the fermenter vessel with fresh perfusion media and retentate recombinant E. coli for continuous production and secretion.
Expression vector SEQ ID NO: 6 encoding GLP-1, ompf signal peptide, and truncated YebF
The expression vector consists of SEQ ID NO: 6 and includes SEQ ID NO: 1 encoding the recombinant GLP-1 peptide, SEQ ID NO: 3 encoding the secretory signal peptide of the gene ompf, and a DNA sequence encoding a truncated YebF peptide of SEQ ID NO: 5.
Induction triggered by residual glucose/dextrose reduction to approximately 5 g/L
Lac operon inducing is added when residual glucose/dextrose is reduced to approximately 5 g/L for induction of production and secretion of recombinant GLP-1 peptide from recombinant E. coli.
Chemically defined initial batch and perfusion media with pH maintained at 6.9
The perfusion-based fermenter system uses initial batch media and chemically defined perfusion media, with pH maintained at 6.9.
The claim coverage centers on a method combining the SEQ ID NO: 6 expression vector, residual glucose/dextrose-triggered induction, and perfusion-based separation of retentate and permeate to enable continuous production and secretion of recombinant GLP-1 peptide.
Stated Advantages
Enables continuous production and secretion of recombinant GLP-1 peptide.
Provides secretion of recombinant GLP-1 peptide in the range of 1–1.2 g/L/hr by a perfusion-based fermentation system.
Documented Applications
Continuous production and secretion of recombinant GLP-1 peptide using E. coli in a perfusion-based fermentation system.
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