# US 11,440,001 B1 · High-throughput crystallographic screening device and method for crystalizing membrane proteins using a sub physiological resting membrane potential across a lipid matrix of variable composition > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,440,001 B1 - Document ID: US11440001B1 - Status: Granted - Kind: B1 - Application number: 15/996,946 - Filed: 2018-06-04 - Granted: 2022-09-13 - Earliest claimed: 2016-06-29 - Assignees: University of Puerto Rico - Applicants: University of Puerto Rico - Inventors: Jose A. Lasalde-Dominicci; Orestes Quesada-Gonzalez; Josue Rodriguez-Cordero; Carlos Baez-Pagan; Martin Montoya-Zavala - Web page: https://patentgrep.com/patent/US11440001B1 ## CPC - B01L 3/06: Crystallising dishes - B01J 19/0046: {Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays (synthesis methods per se C40B50/00)} - B01J 19/1893: {Membrane reactors (catalytic membranes B01J35/59; membranes B01D71/00)} - B01L 3/5085: {for multiple samples, e.g. microtitration plates} - C07K 1/306: {by crystallization} - C30B 7/12: by electrolysis - C30B 29/58: Macromolecular compounds - C40B 40/10: Libraries containing peptides or polypeptides, or derivatives thereof - B01J 2219/00317: Microwell devices, i.e. having large numbers of wells - B01J 2219/00522: in a multiple parallel arrangement - B01J 2219/00585: Parallel processes - B01J 2219/00639: the compounds being trapped in or bound to a porous medium - B01J 2219/0072: Organic compounds - B01J 2219/00725: Peptides - B01J 2219/00745: Inorganic compounds - B01J 2219/00756: Compositions, e.g. coatings, crystals, formulations - B01L 2300/069: Absorbents; Gels to retain a fluid - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C30B: SINGLE-CRYSTAL GROWTH (by using ultra-high pressure, e.g. for the formation of diamonds, B01J3/06); UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL (zone-refining of metals or alloys C22B); PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE (casting of metals, casting of other substances by the same processes or devices B22D; working of plastics B29; modifying the physical structure of metals or alloys C21D, C22F); SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE (for producing semiconductor devices or parts thereof H10); APPARATUS THEREFOR - C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60) ## Abstract The invention is a high-throughput voltage screening crystallographic device and methodology that uses multiple micro wells and electric circuits capable of assaying different crystallization condition for the same or different proteins of interest at the same of different voltages under a humidity and temperature controlled environment. The protein is solubilized in a lipid matrix similar to the lipid composition of the protein in the native environment to ensure stability of the protein during crystallization. The invention provides a system and method where the protein is transferred to a lipid matrix that holds a resting membrane potential, which reduces the degree of conformational freedom of the protein. The invention overcomes the majority of the difficulties associated with vapor diffusion techniques and essentially reconstitutes the protein in its native lipid environment under “cuasi” physiological conditions. ## Claims ### Claim 1 A system for membrane protein crystallization comprising: a sample unit having: a sample holding layer having a plurality of holding wells, wherein each holding well comprises a pair of electrodes, and a lid layer provided over said plurality of holding wells to seal the contents of said plurality of holding wells from each other; a variable direct current potential source electrically coupled to the pair of electrodes of each holding well; wherein the variable potential source is configured to alter a direction of a potential field generated between the pair of electrodes of each holding well, wherein the direction of the potential field generated oscillates 180 degrees in under 1 second. ### Claim 2 (depends on 1) The system of claim 1, wherein said lid layer comprises a plurality of lid wells having the same shape and dimensions as said holding wells. ### Claim 3 (depends on 1) The system of claim 1, wherein said lid layer comprises an adhesive sheet. ### Claim 4 (depends on 1) The system of claim 1, wherein said variable direct current potential source provides the same variable direct current potential to the pair of electrodes ends of all holding wells. ### Claim 5 (depends on 1) The system of claim 1, wherein said variable direct current potential source provides different variable direct current potentials to the pair of electrodes ends of different holding wells. ### Claim 6 (depends on 1) The system of claim 1, wherein said sample unit comprises at least one membrane protein sample. ### Claim 7 (depends on 6) The system of claim 6, wherein said at least one membrane protein sample comprises solubilized membrane protein complex. ### Claim 8 (depends on 7) The system of claim 7, wherein said at least one membrane protein sample is provided in a lipid matrix. ### Claim 9 (depends on 8) The system of claim 8, wherein said lipid matrix has a lipid composition similar to the lipid composition of the membrane protein in the native environment or is varied by lipid doping. ### Claim 10 (depends on 8) The system of claim 8, wherein pH and ionic content of the lipid matrix is adjusted prior to crystallization. ### Claim 11 (depends on 1) The system of claim 1, wherein said sample unit comprises a plurality of membrane protein samples of the same membrane protein. ### Claim 12 (depends on 1) The system of claim 1, wherein said sample unit comprises a plurality of membrane protein samples of different membrane proteins. ### Claim 13 (depends on 1) The system of claim 1, wherein said potential source provides to the pair of electrodes ends of the holding wells a potential selected from: a subphysiological membrane potential, a physiological membrane potential and a supra-physiological membrane potential. ### Claim 14 (depends on 13) The system of claim 13, wherein said potential source provides to the pair of electrodes ends of the holding wells a physiological membrane potential prior to providing said sub-physiological membrane potential. ### Claim 15 (depends on 1) The system of claim 1, wherein said potential source provides a constant potential. ### Claim 16 (depends on 1) The system of claim 1, wherein said potential source varies a waveform of said potential. ### Claim 17 (depends on 1) The system of claim 1, further comprising an X-ray Diffraction instrument. ### Claim 18 (depends on 1) The system of claim 1, further comprising a Fluorescence Recovery After Photobleaching instrument. ### Claim 19 (depends on 1) The system of claim 1, wherein a temperature unit and a humidity unit control the temperature and humidity conditions during a crystallization process of a membrane protein sample contained within a holding well of said sample unit. ### Claim 20 (depends on 1) The system of claim 1, wherein said potential source comprises a multi potential source. ### Claim 21 (depends on 1) The system of claim 1, wherein at least one of: the potential source, the temperature unit and the humidity unit is external to said incubator. ### Claim 22 (depends on 19) The system of claim 19, wherein at least one of: the potential source, the temperature unit and the humidity unit is integrated into an incubator. ### Claim 23 (depends on 22) The system of claim 22, wherein said incubator receives a plurality of sample units. ### Claim 24 (depends on 1) The system of claim 1, wherein each holding well of the plurality of holding wells has the same geometric shape. ### Claim 25 (depends on 1) The system of claim 1, wherein the pair of electrodes have the same geometric shape in every holding well. ### Claim 26 (depends on 1) The system of claim 1, wherein at least one holding well has a pair of electrodes having a geometric shape different than the pair of electrodes ends of said plurality of holding wells. ### Claim 27 (depends on 1) The system of claim 1, wherein the holding wells have the same volume.