United States of America as Represented by the Secretary of the Air Force

Patrick B Dennis

Rajesh R Naik

Joseph M Slocik

A method for transfecting microorganisms comprises inoculating a growth media consisting of at least one of sterile LB media and tryptic soy broth with microorganism cells (cells) consisting of at least one of(DH5&#x3b1;),orgrowing the cells at between 28-40&#xb0; C. to achieve a desired cell density; harvesting the cells; adding a protein ionic liquid consisting of at least one of green fluorescent protein (GFP), ferritin, rabbit IgG antibodies, and photosystem II from spinach ionic liquid to the cells; suspending the cells in the protein ionic liquid; freezing the suspended cells between &#x2212;20 to &#x2212;212&#xb0; C.; and removing at least 99% of water from the frozen suspended cells to make a cell powder. The cell powder may be reconstituted in Tris HCl buffer and mixed to obtain uniform cell suspension; and centrifuged to obtain cell pellet.

Intracellular non-genetic modification of microorganisms using protein ionic liquids

A method comprising: providing aqueous antibodies; cationizing the aqueous antibodies by the addition of stoichiometric amounts of an excess of a positively-charged crosslinker in the presence of a coupling reagent; titrating the cationized antibodies with a counter anionic polymer until the antibody cation/anion pair solution becomes negative by zeta potential measurement, to create at least one antibody cation/anion pair in aqueous solution. The antibodies are one of anti-hemoglobin antibodies, anti-horse spleen ferritin IgG antibodies, or blood-typing IgM Anti-A antibodies, single-chain antibodies from camelids, monoclonal Anti-Flag antibodies, monoclonal Anti-HRP2 to, polyclonal Anti-neuropeptide Y, and polyclonal Anti-human troponin. The antibody cation/anion pair solution may be lyophilized to remove all of the water, forming a lyophilized solid, and the lyophilized solid may be heated to generate an antibody ionic liquid.

Ultra-stable protein ionic liquids

A method of forming a stable protein complex comprising: providing aqueous protein complexes, wherein the protein complexes are one or more of photosystem I complex from spinach, photosystem II complex from spinach, chlorophyll antennae, thylakoids, bacteriochlorophylls, chlorosomes, and photosystems from green algae, cyanobacteria, and plants; cationizing the aqueous protein complexes by the addition of stoichiometric amounts of a crosslinker in the presence of a coupling reagent; titrating the cationized protein complexes with a counter anionic polymer until the protein cation/anion pair solution becomes negative by zeta potential measurement, to create at least one antibody cation/anion pair in aqueous solution. The protein complexes cation/anion pair solution may be lyophilized to remove all of the water, forming a lyophilized solid. The lyophilized solid may be heated until a protein complex ionic liquid is generated. The cationized protein complexes may be purified from excess coupling reagents by dialysis in water.

A method for creating a stable protein/antibody ionic liquid, comprising: (a) cationizing aqueous proteins/antibodies by addition of an excess of a positively-charged crosslinker in the presence of a coupling reagent; (b) purifying the cationized proteins/antibodies; (c) titrating the cationized proteins/antibodies with a corresponding biologically-compatible counter anionic polymer to create at least one protein/antibody cation/anion pair in aqueous solution until the cation/anion pair solution becomes negative by zeta potential measurement; (d) repeatedly dialyzing the protein/antibody cation/anion pair in water to remove excess anionic polymer using at least one molecular weight cutoff 7000 dialysis membrane; (e) lyophilizing the protein/antibody cation/anion pair to remove most of the water, forming a lyophilized solid; and (f) heating the lyophilized solid until a protein/antibody ionic liquid is generated. The antibody may be any desired antibody, and the anion may be any biologically-compatible anion.

Ultrastable antibody ionic liquids

A method comprising: providing aqueous insulin; titrating the aqueous insulin with a mixture of small molecule anions, e.g. D- and L-amino acid esters, small D- and L-peptide pairs, and DL lactate solution, to form an insulin/anion pair solution. Titrating may be performed until the insulin/anion pair solution becomes negative by zeta potential measurement. The insulin/anion pair solution may be dialyzed to remove excess anionic polymer using a membrane sufficient to separate the insulin/anion pairs from excess small molecule anions. The insulin/anion pair solution may be dialyzed or lyophilized to remove all of the water, forming a solid of ultra-stable insulin. The positive electrostatic charge of the aqueous insulin may be confirmed by measuring a positive zeta potential value.

A method for creating an antibody ionic liquid, comprising: providing aqueous anti-A, anti-B, or anti-Rh antibodies; cationizing the aqueous antibodies by the addition of stoichiometric amounts of an excess of a positively-charged crosslinker in the presence of a coupling reagent; titrating the cationized antibodies with a counter anionic polymer to create at least one antibody cation/anion pair in aqueous solution until the antibody cation/anion pair solution becomes negative by zeta potential measurement. The method may further comprise lyophilizing the antibody cation/anion pair solution to remove all of the water, forming a lyophilized solid; and heating the lyophilized solid to generate a protein ionic liquid. Cationized antibodies may be purified of excess coupling reagents by dialysis in water by a membrane, and the antibody cation/anion solution may be dialyzed to remove excess anionic polymer with a similar membrane.

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.

A stable protein ionic liquid, comprising an anti-hemoglobin cation/anion pair. The anti-hemoglobin cation/anion pair may be an anionic polymer of poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether. The anti-hemoglobin cation/anion pair may further comprise a cationized anti-hemoglobin antibody, single-chain antibodies from camelids, antibody fragments, polyclonal Anti-horse spleen ferritin antibodies, monoclonal Anti-Flag antibodies, monoclonal Anti-HRP2 to, polyclonal Anti-neuropeptide Y, polyclonal Anti-human troponin, isotypes of antibodies, or combinations of multiple antibodies.

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Cincinnati, OH, United States of America

Patrick B Dennis