Ultra-stable protein ionic liquids
Inventors
Slocik, Joseph M • Naik, Rajesh R. • Dennis, Patrick B
Assignees
United States Department of the Air Force
Publication Number
US-11274289-B1
Publication Date
2022-03-15
Expiration Date
2037-02-23
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Abstract
A method comprises the steps of: providing aqueous enzymes, wherein the enzymes are one of BamH1, EcoR1, EcoR2, and EcoRV; titrating the aqueous enzymes with a mixture of small molecule anions to form an enzyme/anion pair solution. Small molecule anions may comprise one or more of D- and L-amino acid esters, small D- and L-peptide pairs, and DL lactate solution. The titrating step is performed until the enzyme/anion pair solution becomes negative by zeta potential measurement. The at least one enzyme/anion pair may be dialyzed to remove excess anionic polymer using a dialysis membrane. The enzyme/anion pair solution is lyophilized to remove all of the water, forming a lyophilized solid of ultra-stable enzymes. Before titration, the positive electrostatic charge of the aqueous enzymes may be confirmed by measuring a positive zeta potential value.
Core Innovation
The invention provides a method for creating ultra-stable, water-free biological materials in the form of protein ionic liquids. It involves cationizing aqueous proteins or antibodies using positively charged crosslinkers and coupling agents to modify negative sites on the proteins, followed by titrating with biologically compatible anions to form protein cation/anion pairs. After removing water through lyophilization and optionally dialysis, heating the lyophilized solid produces a viscous, clear protein ionic liquid that retains biological activity and is stable at elevated temperatures without requiring refrigeration.
The method is applicable to various proteins and antibodies, including IgG, IgY, and others, as well as restriction enzymes like BamH1, EcoR1, EcoR2, and EcoRV without needing cationization due to their native positive charge. The enzyme ionic liquids are formed by titrating aqueous enzymes with small molecule anions until the complex becomes negatively charged by zeta potential. These enzyme ionic liquids are also lyophilized and heated to produce stable, biologically active liquids that maintain enzyme function at temperatures higher than conventional aqueous solutions.
The invention addresses the problem of protein and antibody instability in aqueous solutions, where water promotes hydrolysis, oxidation, denaturation, and reduced shelf life, necessitating permanent refrigeration. Current storage methods require cold supply chains which are costly and impractical, especially in regions lacking electricity. By removing water and converting proteins and enzymes into ionic liquids that maintain biological recognition and activity at elevated temperatures and ambient conditions, the invention aims to eliminate refrigeration needs, extend shelf life, and reduce degradation.
Claims Coverage
The claims include one independent claim focusing on a method for forming ultra-stable enzyme ionic liquids involving enzymes and small molecule anions. The main inventive features are centered on enzyme selection, anion titration, purification, lyophilization, and formation of enzyme ionic liquids.
Method of forming enzyme ionic liquids using specific enzymes and anions
The method comprises providing aqueous enzymes selected from BamH1, EcoR1, EcoR2, and EcoRV, and titrating the enzymes with a mixture of small molecule anions consisting of a DL lactate solution to form enzyme/anion pairs.
Control of charge state by zeta potential measurement
Titration is performed until the enzyme/anion pair solution reaches a negative zeta potential below 0 mV to about −1 mV, ensuring a minor excess of anion and charge balance.
Purification by dialysis using molecular weight cutoff membranes
The method optionally includes dialyzing the enzyme/anion pairs with membranes having molecular weight cutoffs between 6000 and 15,000 g/mol to remove excess anionic polymers or small molecules.
Water removal by lyophilization
Lyophilizing the enzyme/anion pair solution removes all or most of the water to produce a lyophilized solid ultra-stable enzyme material.
Heating lyophilized solids to produce viscous enzyme ionic liquids
Heating the lyophilized solid over at least 20 minutes at about 40-90° C., or approximately 50° C., generates a viscous, clear enzyme ionic liquid without compromising enzyme activity.
Testing enzymatic activity post-stabilization
The enzyme ionic liquids are tested for functionality by exposing them to double-stranded DNA and assessing cleavage through DNA fragment observation and gel electrophoresis.
The claims cover a method for producing ultra-stable enzyme ionic liquids from specific enzymes by controlled titration with DL lactate anions, purification, lyophilization, and heating to yield stable liquid enzymes that retain cleavage activity at elevated temperatures.
Stated Advantages
The resulting protein and enzyme ionic liquids are ultra-stable with long shelf lives, exceeding five years.
They do not require refrigeration for storage, handling, or use, eliminating cold supply chain dependence.
The ionic liquids are resistant to extreme temperatures, with stability demonstrated beyond 100° C. and up to 200° C. for antibodies.
They maintain biological recognition and activity comparable to native proteins or enzymes, preserving function after heat exposure.
The technology reduces costs and logistical burdens associated with refrigeration and heavy cooling equipment.
Documented Applications
Refrigeration-free storage and handling of antibodies and enzymes for medical and research use.
Use of antibody ionic liquids in diagnostics, including blood typing detectable with IR active dyes and night vision goggles.
Applications in lateral flow assays, enzyme-linked immunosorbent assays (ELISA), anti-venom/anti-toxin therapeutics, immunotherapy, vaccines, and anti-viral treatments.
Stabilized enzymes used in molecular biology, such as restriction enzymes for DNA cleavage including Gibson assembly.
Potential applications in detection of chemical, biological, nuclear, environmental, and radioactive agents.
Stabilization of various antibody types including IgG, IgY, IgM, and antibody fragments.
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