Host cells and systems for membrane protein expression

Patent metadata

Field	Value
Publication number	US-11319530-B2
Application number	US-201815977788-A
Country	US
Kind code	B2
Filing date	May 11, 2018
Priority date	Nov 13, 2015
Publication date	May 3, 2022
Grant date	May 3, 2022

Abstract

Provided herein are host cells or host cellular expression systems that express a membrane protein. Also, methods are provided that use such host cells or host cellular expression systems to produce higher amounts of the membrane proteins. Further, the cells or cellular systems can be used as tools for the functional characterization of membrane proteins, as well as for screening and drug discovery efforts targeting membrane proteins.

First claim

What is claimed is:

1. A host cell comprising:
an exogenous nucleic acid molecule encoding a membrane protein, said membrane protein comprises at least one mutation; and
wherein the host cell comprises a double mutation at T362I and T373I, or a double mutation at T362I and G512S, in the gene yidC.

2. The host cell of claim 1 , wherein said host cell is a bacterial host cell.

3. The host cell of claim 1 , wherein said host cell is a Escherichia coli.

4. The host cell of claim 1 , wherein the membrane protein is selected from the group consisting of vitamin K epoxide reductase (VKORc1), proteorhodopsin, G protein couple receptors, growth factor receptors, transmembrane ion channels, neurotransmitter transporters, serotonin and olfactory receptors, glycoprotein hormone receptors, chemokine receptors, adenosine receptors, biogenic amine receptors, melanocortin receptors, neuropeptide receptors, chemotactic receptors, somatostatin receptors, opioid receptors, melatonin receptors, calcitonin receptors, parathyroid hormone/parathyroid related protein (PTH/PTHrP) receptors, glucagon receptors, secretin receptors, latrotoxin receptors, metabotropic glutamate receptors, calcium receptors, gamma-aminobutyric acid B (GABA-B) receptors, pheromone receptors, histamine receptors, protease-activated receptors, rhodopsins, C—X—C Motif Chemokine Receptor 4 (CXCR4), G Protein-Coupled Receptor 3 (GPR3), vasopressin receptor, β1 adrenergic receptor, β2 adrenergic receptor, β3 adrenergic receptor, α1 adrenergic receptors and the α2 adrenergic receptor, M1 muscarinic receptor, M2 muscarinic receptor, M3 muscarinic receptor, M4 muscarinic receptor, M5 muscarinic receptor, and angiotensin II receptors.

5. The host cell of claim 4 , wherein the membrane protein is VKORc1 and VKORc1 comprises a mutation having a deletion of amino acid 31-33 (AAR) (VKORc1 ΔAAR ).

6. The host cell of claim 4 , wherein the membrane protein is VKORc1 and VKORc1 comprises a mutation at a residue selected from the group consisting of glycine 60 to aspartic acid (G60D), cysteine 43 to alanine (C43A), cysteine 51 to alanine (C51A), cysteine 132 to alanine (C132A), valine 29 to glutamic acid (V29E), valine 29 to lysine (V29K), arginine 33 to proline (R33P), alanine 48 to serine (A48S), arginine 53 to histidine (R53H), phenylalanine 55 to serine (F55S), serine 57 to proline (S57P), serine 57 to phenylalanine (S57F), arginine 58 to histidine (R58H), serine 52 to asparagine (S52N), arginine 61 to histidine (R61H), glycine 62 to aspartic acid (G62D), glycine 64 to aspartic acid (G64D), threonine 4 to isoleucine (T4I), leucine 65 to proline (L65P), asparagine 80 to aspartic acid (N80D), threonine 137 to proline (T137P), or combination thereof.

7. The host cell of claim 4 , wherein the membrane protein is VKORc1 and VKORc1 comprises two mutations G6OD and VKORc1 ΔAAR .

8. A cell culture comprising the host cell of claim 1 .

9. A method of ligand screening, drug screening, protein capturing, protein purification, immunization, or a biophysical study, wherein the improvement comprises: utilizing the host cell of claim 1 , for ligand screening, drug screening, protein capturing and purification, immunization, biophysical studies.

10. A host cell comprising:
an exogenous nucleic acid molecule encoding a membrane protein, wherein said membrane protein comprises at least one mutation, wherein the membrane protein is vitamin K epoxide reductase (VKORc1); and
wherein the host cell comprises a double mutation at T362I and T373I, or a double mutation at T362I and G512S, in the gene yidC.

11. The host cell of claim 10 , wherein VKORc1 comprises the mutation VKORc1 ΔAAR .

Assignees

• Harvard College

Inventors

• Beckwith Jonathan
• Boyd Dana
• Blazyk Jessica
• Hatahet Feras

Classifications

• C12N1/205

  Bacterial isolates · CPC title

• C07K14/245

  Escherichia (G) · CPC title

• C12N9/90

  Isomerases (5.) · CPC title

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title