E. coli-based production of beta-lactamase
Inventors
Bristol, Andrew • Kaleko, Michael • Hubert, Steven
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11981899-B2
Publication Date
2024-05-14
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 disclosed invention relates to a method for the production of a beta-lactamase polypeptide in Escherichia coli (E. coli). The method provides an E. coli cell transformed with an expression vector comprising a sequence encoding the beta-lactamase polypeptide and induces expression of the beta-lactamase polypeptide in the cytoplasm.
Expression is induced by adding isopropylthiogalactoside (IPTG) to the culture, and the beta-lactamase polypeptide is recovered from a cytoplasmic soluble fraction prepared from the E. coli cell. Recovery or purification is not performed from a periplasmic fraction, a cell envelope fraction, a cytoplasmic insoluble fraction, an inclusion body, or an extracellular fraction.
The disclosure emphasizes high-yield cytoplasmic expression with recovery from cytoplasmic soluble fractions. Documented variants include beta-lactamase polypeptides with defined mutation sets, including Ambler-position substitutions, and examples reporting intracellular fermentation performance, soluble protein predominance, and improved purification outcomes, including a cGMP production run with gram-scale recovery and single-column purification.
Claims Coverage
The independent claim in this family defines a cytoplasmic IPTG-induced production workflow for a beta-lactamase polypeptide in E. coli with explicit exclusions for non-cytoplasmic and non-soluble recovery locations, and dependent claims add quantitative yield thresholds, specific host strains, expression-vector features, and a single chromatography column step. The independent claim is supported by multiple inventive features that together control where expression occurs and where the polypeptide is recovered and purified.
Cytoplasmic induction in transformed E. coli with IPTG
A method for producing a beta-lactamase polypeptide in Escherichia coli using an expression vector encoding the beta-lactamase polypeptide, culturing the E. coli cell to induce expression in the cytoplasm, and inducing expression by adding isopropylthiogalactoside (IPTG) to the culture.
Recovery from cytoplasmic soluble fraction excluding periplasm and envelope fractions
Recovering the beta-lactamase polypeptide from a cytoplasmic soluble fraction prepared from the E. coli cell while not recovering or purifying from a periplasmic fraction, a cell envelope fraction, a cytoplasmic insoluble fraction, an inclusion body, or an extracellular fraction.
High-yield production over 10 grams per liter
The method is limited such that production yields more than 10 grams of the beta-lactamase polypeptide per liter of culture.
Use of BL21(DE3) or W3110 host
The method further specifies that the E. coli cell is selected from BL21(DE3) or W3110.
Expression vector with palindromic DNA looping
The method further specifies that the expression vector contains palindromic DNA looping.
Expression vector suitable for tightly controlled gene expression
The method further specifies that the expression vector is suitable for tightly controlled gene expression.
Single chromatography column step
The method is limited such that the production comprises a single chromatography column step.
Across the independent claim and its dependents, the main inventive theme is cytoplasmic IPTG-induced expression in a transformed E. coli host, paired with recovery from a cytoplasmic soluble fraction while excluding recovery or purification from periplasmic, envelope, insoluble or inclusion body, and extracellular fractions, with optional dependent limitations directed to high yield, particular E. coli strains, expression-vector features, and a single chromatography column purification constraint.
Stated Advantages
High-yield cytoplasmic expression with recovery from cytoplasmic soluble fractions.
Improved purification outcomes.
Gram-scale recovery in a cGMP production run.
Soluble protein predominance.
Documented Applications
Intracellular fermentation performance.
cGMP production run.
Single-column purification.
Interested in licensing this patent?