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

Patent metadata

Field	Value
Publication number	US-2022274101-A1
Application number	US-202217741595-A
Country	US
Kind code	A1
Filing date	May 11, 2022
Priority date	Jun 29, 2016
Publication date	Sep 1, 2022
Grant date	—

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.

First claim

What is claimed is:

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; and
a variable direct current potential source electrically coupled to the pair of electrodes of each holding well.

2 . The system of claim 1 , further comprising:
an incubator receiving the sample unit; a temperature unit monitoring and controlling a temperature inside the incubator; and a humidity unit monitoring and controlling a humidity inside said incubator.

3 . The system of claim 1 , 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 varies within 180 degrees.

4 . The system of claim 1 , wherein the plurality of holding wells has the same geometric shape or different geometric shapes.

5 . 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.

6 . The system of claim 1 , wherein the pair of electrodes has the same geometric shape in every holding well.

7 . 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.

8 . 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.

9 . 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.

10 . The system of claim 1 , wherein said sample unit comprises at least one membrane protein sample, and wherein said at least one membrane protein sample comprises solubilized membrane protein complex.

11 . The system of claim 10 , wherein said at least one membrane protein sample is provided in a lipid matrix.

12 . The system of claim 11 , 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.

13 . The system of claim 1 , wherein said sample unit comprises a plurality of membrane protein samples of the same membrane protein.

14 . The system of claim 1 , wherein said sample unit comprises a plurality of membrane protein samples of different membrane proteins.

15 . 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.

16 . The system of claim 1 , wherein said potential source varies a waveform of said potential.

17 . 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 the intensity of a potential field generated between the pair of electrodes of each holding well.

18 . The system of claim 17 , wherein the alteration in intensity of a potential field occurs at a range of potential from −140 mV to 10 mV.

19 . The system of claim 17 , 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 varies within 180 degrees.

20 . The system of claim 17 , 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.

21 . The system of claim 20 , wherein at least one of: the potential source, the temperature unit and the humidity unit is integrated into an incubator.

22 . The system of claim 17 , wherein said lid layer comprises a plurality of lid wells having the same shape and dimensions as said holding wells.

23 . The system of claim 17 , wherein said variable direct current potential source provides the same variable direct current potential to the pair of electrodes ends of all holding wells.

24 . The system of claim 18 , wherein said sample unit comprises at least one membrane protein sample, and wherein said at least one membrane protein sample comprises solubilized membrane protein complex.

25 . 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 the intensity of a potential field generated between the pair of electrodes of each holding well more than one hour after the first variation in the potential field.

26 . The system of claim 25 , wherein the potential is altered to a range from −5 mV to −20 mV after 1-2 hours in a range from −140 mV to 10 mV.

27 . The system of claim 25 , 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 varies within 180 degrees.

28 . The system of claim 25 , wherein said sample unit comprises a plurality of membrane protein samples of the same membrane protein.

29 . The system of claim 25 , wherein said sample unit comprises a plurality of membrane protein samples of different membrane proteins.

30 . The system of claim 25 , 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.

31 . The system of claim 25 , wherein said sample unit comprises at least one membrane protein sample, and wherein said at least one membrane protein sample comprises solubilized membrane protein complex.

Assignees

• Univ Puerto Rico

Inventors

• Lasalde-Dominicci Jose
• Quesada-Gonzalez Orestes
• Rodriguez-Cordero Josue
• Baez-Pagan Carlos
• Montoya-Zavala Martin

Classifications

• B01J2219/0072

  Organic compounds · CPC title

• B01J19/0046 Primary

  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) · CPC title

• C07K1/306

  by crystallization · CPC title

• B01J19/1893

  Membrane reactors (catalytic membranes B01J35/59; membranes B01D71/00) · CPC title

• C40B40/10

  Libraries containing peptides or polypeptides, or derivatives thereof · CPC title