TECHNICAL FIELD

The disclosure relates to the field of protein expression technologies. More specifically, host cells or host cellular expression systems are provided that express a foreign membrane protein in the host cell. 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 structural and functional characterization of membrane proteins, as well as for screening and drug discovery efforts targeting membrane proteins.

BACKGROUND

While proteins localized to biological membranes constitute about a quarter of the products of an organism's open reading frames, only 0.1% of known protein structures are membrane proteins (1, 2). The lag in our knowledge of the physiology and structural biology of membrane proteins is due to many factors including the low expression levels of membrane proteins and the complex biophysical nature of their interactions at the water/lipid interface. Membrane proteins, which have one or more segments that traverse the lipid bilayer, require dedicated cellular machineries for their targeting to and integration into the membranes (3). Even though these cellular machineries have maintained conserved features in all domains of life, efforts to express membrane proteins in foreign organisms have often been unsuccessful. In many cases, the overexpression of indigenous membrane proteins leads to mis-targeting and inclusion body formation, protein degradation or cell death (4).

Promoting the correct orientation of proteins within the membrane provides an additional challenge to the insertion machinery. Proper assembly is a crucial step in the folding pathway of a membrane protein since a wrong orientation could be detrimental to functioning of the protein and to the viability of the cell. The overall architecture of a membrane protein, including its final membrane topology, is achieved by topogenic signals embedded in the amino acid sequence. Such signals include long stretches of mostly hydrophobic amino acids (around 20) that determine the portion of the protein to be embedded in the membrane. Furthermore, the abundance of positively-charged amino acids in cytoplasmic domains of membrane proteins, generally referred to as the positive-inside rule, is a major contributor to membrane protein topology (5,6).

Most membrane proteins express at low levels in non-engineered eukaryotic or bacterial cells. Different techniques to increase the expression levels of the membrane protein have been taken to enhance membrane protein surface expression in heterologous cells, including addition/deletion of receptor sequences, co-expression with interacting proteins, and treatment with pharmacological chaperones (reviewed in Dunham and Hall, 2009, Trends Biotechnol. 27:541). It remains a challenge, however, to significantly improve total yield, conformational stability and/or functionality of expressed membrane protein.

Thus, it would be advantageous to have alternative expression systems that permit heterologous expression of membrane proteins for isolation, purification, and structural studies. Likewise, it would be advantageous to have membrane proteins in a functional form for screening assays targeting membrane proteins. This would greatly facilitate the whole trajectory of drug discovery efforts on membrane proteins as therapeutic targets.

SUMMARY

Proteins embedded in biological membranes are important targets for therapeutic intervention. However, they tend to be far more challenging to deal with than soluble proteins. Provided herein is a host cell (e.g.,E. coli) useful for the high-level expression of exogenous (e.g., foreign) membrane proteins (e.g., vitamin K epoxide reductase (VKORc1) in functional form.

Thus, one aspect of the invention relates to a host cell comprising: an exogenous nucleic acid molecule encoding a membrane protein (e.g., vitamin K epoxide reductase (VKORc1), said membrane protein comprises at least one mutation (e.g., VKORc1ΔAAR); and at least one host cell-specific mutation in gene selected from a disulfide bond enzyme (e.g., dsbB or dsbA), insertase (e.g., yidC), ubiquinone biosynthesis gene (e.g., ubiC or ubiE), menaquinone biosynthesis gene (e.g., menA), or cytoplasmic protease (e.g., hsIV or hsIU), or combinations thereof.

In some embodiments, the host cell is a bacterial host cell.

In some embodiments, the host cell is aEscherichia coli.

In some embodiments, the nucleic acid molecule is derived from a mouse, rat, rabbit, hamster, gerbil, dog, cat, goat, pig, cow, horse, whale, monkey, or human.

In some embodiments, the membrane protein has at least two, three, four, five, or six mutations.

In some embodiments, the host cell has at least two, three, four, five, or six host cell-specific mutations.

In some embodiments, the mutation is a deletion, substitution, or insertion.

In some embodiments, 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, PTH/PTHrP receptors, glucagon receptors, secretin receptors, latrotoxin receptors, metabotropic glutamate receptors, calcium receptors, GABA-B receptors, pheromone receptors, histamine receptors, protease-activated receptors, rhodopsins, CXCR4, GPR3, rhodopsin, 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.

In some embodiments, the membrane protein is VKORc1.

In some embodiments, the VKORc1 comprises a mutation having a deletion of amino acid 31-33 (AAR) (VKORc1ΔAAR).

In some embodiments, the 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 (C33A), valine 29 to glutamic acid (V29E), valine 29 to lysine (V29K), arginine 33 to proline (A33P), 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.

In some embodiments, the VKORc1 comprises two mutations G60D and VKORc1ΔAAR.

In some embodiments, the at least one host cell-specific mutation is a mutation of at least one gene selected from the group consisting of a disulfide bond enzyme, insertase, ubiquinone biosynthesis gene, menaquinone biosynthesis gene, and cytoplasmic protease.

In some embodiments, the disulfide bond enzyme is dsbB or dsbA.

In some embodiments, the insertase is yidC.

In some embodiments, the cytoplasmic protease is hsIV or hsIU.

In some embodiments, the ubiquinone biosynthesis gene is selected from ubiC or ubiE.

In some embodiments, the menaquinone biosynthesis gene is menA.

In some embodiments, the yidC comprises a mutation at a residue selected from the group consisting of alanine 11 to threonine (A11T), threonine 362 to isoleucine (T362I), threonine 362 to alanine (T362A), threonine 362 to serine (T362S), threonine 362 to phenylalanine (T362F), threonine 362 to valine (T362V), threonine 362 to leucine (T362L), threonine 362 to glutamic acid (T362E), threonine 362 to arginine (T362R), threonine 362 to asparagine (T362N), threonine 362 to aspartic acid (T362D), threonine 362 to cysteine (T362C), threonine 362 to glutamine (T362Q), threonine 362 to glycine (T362G), threonine 362 to histidine (T362H), threonine 362 to methionine (T362M), threonine 362 to proline (T362P), threonine 362 to tryptophan (T362W), threonine 362 to tyrosine (T362Y), threonine 362 to lysine (T362K), arginine 366 to isoleucine (R366I), threonine 373 to isoleucine (T373I), glutamine 429 to isoleucine (Q429I), threonine 474 to isoleucine (T474I), glycine 512 to serine (G512S), serine 520 to isoleucine (S520I), asparagine 521 to isoleucine (N521I), and glutamine 527 to isoleucine (Q527I), or combination thereof.

In some embodiments, the yidC comprises a mutation at T362I.

In some embodiments, the yidC comprises a double mutation at T362I and T373I.

In some embodiments, the yidC comprises a double mutation at T362I and G512S.

In some embodiments, the yidC comprises a mutation at T362D, T362C, T362Q, T362G, T362H, T362M, T362P, T362W, T362Y, T362N, or T362R supplemented with a plasmid coding for YidCWT.

In some embodiments, the hsIV comprises a mutation at residue cysteine 160 to tyrosine (C160Y) or threonine 163 to isoleucine (T163I).

In some embodiments, the mutation is a deletion of hsIV or hsIU.

In some embodiments, the mutation is a deletion of dsbB or dsbA.

In some embodiments, the dsbA comprises a mutation at residue cysteine 33 to alanine (C33A).

In some embodiments, the mutation is a deletion of menA

In some embodiments, the host cell further comprises a reporter.

In some embodiments, the reporter is β-galactosidase.

In some embodiments, the β-galactocidase is a disulfide-bond sensitive enzyme.

In some embodiments, the disulfide-bond sensitive enzyme is β-galdbs.

In some embodiments, the exogenous nucleic acid molecule is under the control of a promoter.

In some embodiments, the promoter is a constitutive promoter or an inducible promoter.

In some embodiments, the inducible promoter is an isopropyl β-D thiogalactopyranoside (IPTG)-inducible promoter.

A cell culture of the host cell is also provided. Another aspect of the invention relates to a polypeptide expressed by said host cell or a membrane preparation of said host cell. Another aspect of the invention relates a method of inhibitor, ligand screening, drug screening, protein capturing, protein purification, immunization, or a biophysical study, wherein the improvement comprises: utilizing said host cell, for inhibitor, ligand screening, drug screening, protein capturing and purification, immunization, biophysical studies.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description

