E. coli-based production of beta-lactamase
Inventors
Bristol, Andrew • Kaleko, Michael • Hubert, Steven
Assignees
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Publication Number
US-11542510-B2
Publication Date
2023-01-03
Expiration Date
Abstract
The invention relates to, in part, improved methods for the production of beta-lactamase using Escherichia coli (E. coli) cells. High yield production of beta-lactamase is achieved using methods of the invention.
Core Innovation
The invention relates to methods for the production of a beta-lactamase polypeptide in Escherichia coli (E. coli). The methods provide an E. coli cell transformed with an expression vector comprising a sequence encoding the beta-lactamase polypeptide, and culture is used to induce expression in the cytoplasm. The produced beta-lactamase polypeptide is recovered from a cytoplasmic soluble fraction prepared from the E. coli cell.
A central aspect is that the beta-lactamase polypeptide is not recovered or purified from a periplasmic fraction, a cell envelope fraction, a cytoplasmic insoluble fraction, an inclusion body, or an extracellular fraction of the E. coli cell. The approach is described as achieving production yields of more than 10 grams per liter of culture, including examples yielding more than 15 grams per liter of culture.
The document further describes scope for beta-lactamase sequence definitions and variant relationships, including polypeptides and polynucleotides associated with SEQ ID NOs and identity thresholds. It also describes producing and purifying beta-lactamase with a large-scale cGMP manufacturing approach, including a single chromatography purification step and reporting high purity recovery.
Claims Coverage
The partial content identifies two independent claims: clm-00001 and clm-00014. Across these claims, the coverage centers on cytoplasmic expression and recovery from a cytoplasmic soluble fraction while expressly excluding recovery/purification from periplasmic, cell envelope, cytoplasmic insoluble, inclusion body, and extracellular fractions, together with quantitative yield thresholds.
Cytoplasmic expression and cytoplasmic soluble recovery in E. coli
A method for the production of a beta-lactamase polypeptide in Escherichia coli comprising providing an E. coli cell transformed with an expression vector comprising a sequence encoding the beta-lactamase polypeptide, culturing to induce expression of the beta-lactamase polypeptide in the cytoplasm, and recovering the beta-lactamase polypeptide from a cytoplasmic soluble fraction prepared from the E. coli cell.
Yield exceeding 10 grams per liter with exclusion of multiple fraction sources
The method yields more than 10 grams of the beta-lactamase polypeptide per liter of culture, and the beta-lactamase polypeptide is not recovered or purified from a periplasmic fraction, a cell envelope fraction, a cytoplasmic insoluble fraction, an inclusion body, or an extracellular fraction of the E. coli cell.
Yield exceeding 15 grams per liter with exclusion of multiple fraction sources
A method for the production of a beta-lactamase polypeptide in Escherichia coli comprising providing an E. coli cell transformed with an expression vector comprising a sequence encoding the beta-lactamase polypeptide, culturing to induce expression of the beta-lactamase polypeptide in the cytoplasm, recovering the beta-lactamase polypeptide from a soluble fraction prepared from the E. coli cell, wherein the method yields more than 15 grams of the beta-lactamase polypeptide per liter of culture, and wherein the beta-lactamase polypeptide is not recovered or purified from a periplasmic fraction, a cell envelope fraction, a cytoplasmic insoluble fraction, an inclusion body, or an extracellular fraction of the E. coli cell.
The claim coverage is directed to producing a beta-lactamase polypeptide in E. coli by inducing cytoplasmic expression from a transformed host and recovering the polypeptide from a cytoplasmic soluble fraction, while expressly excluding recovery/purification from periplasmic, cell envelope, cytoplasmic insoluble, inclusion body, or extracellular fractions. The independent claims impose quantitative yield thresholds of more than 10 g/L or more than 15 g/L.
Stated Advantages
High production yield, including more than 10 grams per liter of culture and more than 15 grams per liter of culture.
Recovery and purification focused on cytoplasmic soluble fraction, with explicit avoidance of recovery/purification from periplasmic, cell envelope, cytoplasmic insoluble, inclusion body, and extracellular fractions.
High purity recovery associated with a single chromatography purification step in the described cGMP manufacturing approach.
Documented Applications
Production and purification of beta-lactamase polypeptide in a large-scale cGMP manufacturing context.
Use of produced beta-lactamase polypeptide associated with therapeutic/microbiome-protecting use is referenced in the partial content summary.
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