JP2003512060A - Expression system for membrane proteins - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for preparing a polypeptide, comprising: a host cell for using the polypeptide for protein expression, wherein the host cell has an altered membrane composition; Expressing in a host cell, isolating a membrane fraction from the host cell, and isolating the polypeptide from the membrane fraction.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to expression systems for polypeptides. In particular, the present invention relates to host cells for expressing polypeptides with modified membrane organization.

[0002] The over-expression of a number of proteins in the background of the invention E. coli (E.coli) is impair the growth of E. coli,
Furthermore, it sometimes kills bacteria. These toxic effects are especially severe with membrane proteins. One of the obstacles to the analysis of membrane proteins is the lack of a generally applicable system for examining membrane protein overexpression (Walker and Saraste, 1996).

Although E. coli is often successful as a delivery vehicle for overexpressing prokaryotic and eukaryotic proteins (see Hockney, 1994), it expresses the majority of membrane proteins and many of the globular proteins. The host bacterium is killed (Dong et al., 1995; Durland and Dong, 1996; Miroux and Walker, 1996).

This problem is particularly relevant in the host Escherichia coli BL21 (DE3) strain, which contains bacteriophage T7 RNA.
It occurs in a widely used bacterial expression system in which the target gene is transcribed by a polymerase (Studier et al., 1990).

To overcome these difficulties with the T7 system, a host of mutants that can overproduce some membrane proteins and some globular proteins that are necessarily toxic when expressed in BL21 (DE3). Were selected from E. coli BL21 (DE3). Such mutant host strains are E. coli C41 (DE3) and C43 (DE3). Lower expression levels in BL21 (DE3) resulted in slightly higher expression levels in C41 (DE3) or C43 (DE3)
(Miroux and Walker, 1996). However, even with these C41 / C43 lines, overexpression with multiple polypeptides remains a problem.

The present invention seeks a method to overcome these problems in conventional protein expression systems.

SUMMARY OF THE INVENTION The present invention relates to protein expression in host cells in which the production of intracytoplasmic membranes has been induced. The intracytoplasmic membranes of the present invention have a modified membrane organization as compared to the membranes of other cells, which is useful for protein expression, particularly polypeptide product isolation. In particular, the invention relates to methods of preparing polypeptides in such host cells.

Thus, in a first aspect, the invention provides a method of producing a polypeptide, which method comprises the steps of: a) a bacterial host containing a membrane in the cytoplasm. Providing a host cell, the cytoplasmic membrane of which has a different structure than the other membranes of the host cell; b) the host cell comprising a coding sequence encoding the polypeptide. Transforming with a nucleic acid construct and expressing the polypeptide in the bacterial host cell such that its expression is associated with a cytoplasmic membrane; and c) the cytoplasmic membrane with other cells. Isolating the polypeptide by separating it from the sex component.

The cytoplasmic membrane architecture has some resistance to protein expression, particularly to the toxic effects of membrane protein expression.

The term “polypeptide” is used interchangeably herein with the term “protein” and means any polypeptide that is two or more amino acid residues in length. Expression of a protein means that the process of transcription (if necessary) and translation produces the polypeptide from the nucleic acid encoding the polypeptide.

The host cell of the present invention may be any cell as long as it can stably or temporarily store the recombinant nucleic acid molecule. Preferably, the host cells of the present invention are bacterial cells such as E. coli cells. Examples of suitable host cells are described in more detail below.

The composition of the cytoplasmic membrane of the present invention is different from that of wild-type cells. The presence of such a difference preferably means a difference in the quality and / or amount of the constituent components of the lipid-containing fraction of the cell. This point will be described in more detail below. "Modified" herein as having different membrane configurations
It is called a film structure. The host cells disclosed herein contain an intracytoplasmic membrane with an altered membrane organization.

The modified membrane configurations of the membranes of the present invention can be characterized by the analytical techniques described below and known to those skilled in the art. Preferably, the membrane of the present invention may have one or more properties selected from the following properties; a lipid: protein ratio of greater than about 0.4; a cardiolipin ratio of greater than about 4%. Phospholipid composition of
Membrane phospholipid composition with a phosphatidyl glycerol ratio of less than about 20%, or membrane phospholipid composition with phosphatidyl ethanolamine levels below average.

The toxic effects of protein expression in host cells can manifest in a wide variety of ways, including delayed growth, reduced viability, morphological defects, or known to those of skill in the art. Other impacts of, and are discussed in more detail below.

It has been shown herein that the toxic effects of protein expression in host cells are partially mitigated by the expression of at least a portion of subunit b or subunit c of ATP synthase. It is further found herein that expression of subunit b or subunit c of ATP synthase makes a surprising contribution to the modification of the membrane organization of the host cells of the invention. Accordingly, the present invention provides for the production of intracytoplasmic membranes induced by the expression of subunit b and / or subunit c of ATP synthase, or a fragment thereof, which is capable of inducing increase of membranes in host cells. It is provided.

Nucleic acid constructs for use in the methods of the invention can be prepared using recombinant DNA techniques known to those of skill in the art and are described below. Briefly, the construct used for protein expression comprises a nucleotide sequence (referred to as the "coding" sequence, or open reading frame (ORF)) that encodes the protein to be expressed, and the coding sequence It may also include a nucleotide sequence (referred to herein as a "promoter") that promotes, directs, or drives the expression of the. The nucleic acid construct may also contain other elements, examples of which are described below.

“ATP synthase subunit b or subunit c” is preferably Escherichia coli A
It is subunit b or subunit c obtained from the F0 membrane region of TP synthase (Walk
er et al., 1982 Nature vol.298, pp867-869). In a highly preferred embodiment, this means subunit b of the F0 membrane region of E. coli ATP synthase or a part thereof, examples of which are mentioned herein.

Expression of a polypeptide of interest in a host cell can be conveniently directed by a nucleic acid construct. Accordingly, the invention provides a host cell comprising a nucleic acid construct, wherein the construct is capable of directing the expression of the polypeptide of interest and at least a portion of subunit b or subunit c of ATP synthase. provide. In a highly preferred embodiment, expression of the polypeptide of interest and expression of at least part of subunit b or subunit c of ATP synthase is directed by a single nucleic acid construct.

The host cell characteristics disclosed herein are particularly advantageous for expression / production of membrane proteins. The membrane protein may be any protein associated with or capable of being associated with the membrane fraction of cells.

As disclosed herein, the production of a membrane having an altered membrane constitution can be induced by expression of at least a portion of subunit b or subunit c of ATP synthase in a host cell. it can. This altered membrane organization can manifest itself in increased production of membranes in the cytoplasm.

Advantageously, the expression of at least a portion of subunit b or subunit c of ATP synthase as well as the expression of the polypeptide of interest is directed from a single nucleic acid construct. It is preferred that expression of at least part of subunit b or subunit c of ATP synthase as well as expression of the polypeptide of interest occur simultaneously. In a preferred embodiment, the polypeptide of interest is a heterologous or integral membrane protein.

By co-expressed is meant that the expression of the polypeptide of interest and the expression of subunit b or subunit c of ATP synthase temporally overlap. In some embodiments, expression of subunit b or subunit c of ATP synthase can precede expression of the protein of interest. In this case, if both proteins are present in the same host cell at a particular time, their expression is considered to overlap.

Advantageously, in the vector of the invention, the coding sequence encoding ATP synthase or a fragment thereof is proximal to the promoter of the coding sequence encoding the polypeptide (
Placed (for example upstream), the two coding sequences are expressed in tandem from the same promoter.

Preferably, the promoter used in the vector of the present invention is an inducible promoter.

Induction of intracellular membrane production is preferably performed prior to or concomitant with production of the polypeptide in the host cell. This ensures that the intracellular membrane is present during polypeptide expression.

The present invention is useful for the expression of polypeptides, which expression is directed by any of the numerous suitable methods known to those of skill in the art and described herein. For example, the expression system can utilize the bacteriophage T7 RNA polymerase system. Accordingly, the present invention provides a method for expressing a protein in a host cell, such that the host cell expresses a bacteriophage RNA polymerase and the expression system contains a promoter sequence recognized by the polymerase. .
Preferably the polymerase is T7 RNA polymerase.

A commonly used system for carrying out such an expression method is pET or pMW.
7 expression systems are included. Therefore, the present invention provides a method for expressing a protein in a host cell, the expression system of which comprises the expression vector pET or pMW7.

It is often desirable to label the expressed protein. Such labeling can be done using any suitable method known to those skilled in the art, for example expressing the polypeptide as a fusion protein with a detectable moiety such as green fluorescent protein (GFP). It can be mentioned. Thus, the invention provides a method for expressing a protein in a host cell, wherein the expression vector comprises a nucleic acid sequence encoding a polypeptide that functions as a detectable label. Preferably the detectable label is green fluorescent protein.

Transformation includes introducing the nucleic acid into a host cell by transfection, infection, transduction, or other means, and also using a non-replicating vector (“suicide vector”) Transfection or transformation as well as stable transformation with either naked nucleic acid or particles carrying nucleic acid are also contemplated. The term vector as used herein refers to a plasmid,
It includes cosmids, gene cassettes, and vectors derived from organisms such as bacteriophage or the like. A control sequence is a nucleic acid sequence that induces, enhances, promotes, or exerts some influence on the expression of the polypeptide of interest.

The polypeptide expressed in the present invention can be isolated by separating the cytoplasmic membrane from other cellular membranes. Preferably, the separation is done by centrifugation. Membrane fractions can be prepared from host cells containing membranes having the modified membrane composition described herein, which method requires the high-speed centrifugation step required by conventional membrane preparation methods. Not. As disclosed herein,
In the present invention, centrifugation at a low speed such as 2,500 to 10,000 × g is sufficient for separating the membrane from the cytoplasmic fraction of host cells. The advantage of this method is that the intracytoplasmic membranes with modified membrane organization are also separated from other cellular membranes, which should sediment at higher rates.

The polypeptide prepared herein can be a membrane protein. In another embodiment, the membrane protein can be a polypeptide fused to a membrane targeting protein.

In a further embodiment of the invention there is provided a method of producing a membrane protein for use in a screening assay. Screening agents for their ability to associate or bind with membrane polypeptides may require preparation and / or expression of the polypeptides. Accordingly, the present invention also relates to a method of screening for agents that bind, affect or modify a desired membrane protein, which method comprises host cells as described herein with a vector of the invention. Transformed
A drug for inducing expression of a desired membrane protein, culturing a host cell to produce the desired membrane protein, and immobilizing a cell membrane on a carrier to screen the membrane Exposing under conditions that promote interactions.

The term agent, as used herein, refers to any agent that is known to or is believed to associate reversibly or irreversibly with a polypeptide, such as a membrane protein. It also means something. Carriers on which cell membranes are immobilized are well known in the art and include activated or coated filters such as nitrocellulose-based filters, supported filters, or ELISA plates. Included surface.

It may be desirable to screen the polypeptides for whole cells without fractionating the host cells during or after expression of the polypeptides of the invention. Accordingly, in a further embodiment, the invention provides a method of screening for agents that bind, affect or modify a desired polypeptide, which method comprises the step of transforming a host cell as described herein. Transforming with the vector of the invention, inducing E. coli F-ATPase subunit b or subunit c to be expressed from the first expression unit and culturing the host cell to induce membrane production. And induce the desired membrane protein to be expressed from the second expression unit,
Culturing the host cell to produce the desired membrane protein, immobilizing the cell on a carrier, and allowing the drug to be screened under conditions that promote the interaction between the drug and the polypeptide Exposure step.

Expression of polypeptides having toxic effects can be improved by culturing the host cells at subnormal temperatures. One common growth temperature for E. coli is 37 ° C.
Is. Accordingly, the present invention relates to a method of expressing a polypeptide in a host cell in which the host cell is cultured at 25 ° C.

DESCRIPTION OF THE INVENTION The term protein means any polypeptide having two or more amino acid residues or longer. The terms protein and polypeptide may be used interchangeably herein and include, inter alia, post-translationally modified polypeptides, or unnatural amino acid or imino acid residues, amino acid analogs, etc. Is included.

Host Cell The host cell of the present invention may be any cell capable of stably or temporarily retaining a nucleic acid molecule capable of inducing the expression of the polypeptide.
Preferably, the host cells of the present invention are bacterial cells such as E. coli cells. More preferably, the host cells of the invention are E. coli C43 (DE3) (ECCC B96070445), E. coli C41 (DE3).
(ECCC B96070444), E. coli DK8 (DE3) S (NCIMB 40885), or E. coli C2014 (DE3
) (NCIMB 40884), or derived therefrom. The term "derived" as used with respect to E. coli strains means that the host cells can be genetically related to one or more strains set forth herein. For example, an E. coli strain is considered to be derived from another strain if the strain was obtained from another strain by basic genetic engineering known to those of skill in the art.

[0038] "toxic" toxic, "toxic" and "toxic effects" are relative terms that can be readily understood in the art. For example, Studier et al. (1990) address the problem of genes that produce products that are toxic to host cells, without requiring a more detailed definition of the term toxic. .

However, toxicity usually manifests itself as a variety of effects on cells. These include impaired cell proliferation, decreased copy number, increased number of cells lacking plasmid in growth medium (Studier et al., 1990), filamentation of bacterial cells (George et al., 1994), induction of SOS response. (Murli and Walker, 1993), and / or ribosome decay (Dong et al.,
1995) is included.

Vectors The nucleic acids of the invention which encode the subject polypeptides can be incorporated into vectors for further manipulation. As used herein, a vector (or plasmid) is a DNA in a cell for expressing or replicating heterologous DNA.
Means an independent element used to introduce Selection and use of such carrier media is within the skill of one in the art. A large number of vectors are available and the selection of the appropriate vector will depend on the intended use of the vector (ie whether it is used for DNA amplification or DNA expression), the size of the DNA that will be inserted into the vector. , And the host cell to be transformed with the vector. Each vector contains various components depending on its functionality (amplification of DNA or expression of DNA) and the host cell to which the vector is compatible. Vector components typically include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, enhancer elements, promoters, transcription termination sequences, and signal sequences.

Both expression and cloning vectors usually contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. In cloning vectors, this sequence usually enables the vector to replicate independently of the host chromosomal DNA, including origins of replication, or autonomously replicating sequences. Such sequences are well known for various bacteria, yeast, and viruses. The origin of replication of plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin of replication is suitable for yeast, and various viral origins of replication (e.g. SV40, polyoma, adenovirus) are used in mammalian cells. Useful as a cloning vector. Usually the origin of replication component is a high-level DNA
It is not required for mammalian expression vectors unless used in mammalian cells, such as COS cells, which are capable of replication.

The majority of expression vectors are shuttle vectors, that is, they are capable of replicating in at least one class of organism, while being transfectable for expression in another class of organism. For example, one vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells. In this case, the vector cannot replicate independently of the host cell chromosome. It can be replicated by inserting the DNA into the host genome. However, the recovery of genomic DNA encoding the polypeptide of interest is more complicated than the recovery of an externally replicated vector because it requires digestion with a restriction enzyme to excise the DNA encoding the polypeptide of interest. . DNA can be amplified by PCR and may be directly transfected into host cells without any replication components.

Selectable Markers Advantageously, the expression and cloning vectors may also include a selectable gene, termed a selectable marker. This gene encodes a protein required for the survival or growth of transformed host cells grown in a selective medium. Host cells not transformed with the vector containing the selection gene will not survive in the medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, or that supply essential nutrients not available from complex media.

As a selection gene marker suitable for yeast, any marker gene can be used as long as it can facilitate the selection of transformants by expressing the phenotype of the marker gene. Suitable markers in yeast include, for example, those that confer resistance to the antibiotics G418, hygromycin, or bleomycin, or those that confer prototrophy to auxotrophic yeast mutants, such as URA3, LEU2,
The LYS2, TRP1, or HIS3 genes may be mentioned.

Since replication of the vector is conveniently carried out in E. coli, it is advantageous to include an E. coli genetic marker and an E. coli origin of replication. These are pBR322, Bluesc
It can be obtained from the ript® vector or an E. coli plasmid containing both the pUC plasmid, eg pUC18 or pUC19, of the E. coli origin of replication and the E. coli genetic marker conferring resistance to antibiotics such as ampicillin.

Promoter expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the polypeptide of interest. Such promoters can be inducible or constitutive. Such a promoter is operably linked to the DNA encoding the polypeptide of interest by removing from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. A large number of heterologous promoters can be used to drive the amplification and / or expression of the polypeptide of interest. The term "operably linked" means that the components are in a relationship such that they can function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

Suitable promoters for use with a prokaryotic host include, for example, β-
Included are hybrid promoters such as the lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and tac promoters. Their nucleotide sequences are publicly available, whereby those skilled in the art will use them to provide DNAs encoding the polypeptide of interest using linkers or adapters to provide any required restriction enzyme cleavage sites.
Can be operatively linked to. Promoters for use in bacterial systems also usually contain a Shine-Dalgarno sequence operably linked to the DNA encoding the polypeptide of interest.

A preferred expression vector is a bacterial expression vector containing a bacteriophage promoter such as phagex or T7 that is functional in bacteria. In one of the most widely used expression systems, the nucleic acid encoding the fusion protein is T7 RNA.
It can be transcribed from the vector using a polymerase (Studier et al., Methods
in Enzymol. 185; 60-89, 1990). When the E. coli BL21 (DE3) host strain is used with the pET vector, T7 RNA polymerase is produced from the host bacterium lambda-lysogen phage DE3 and its expression is under the control of the IPTG-inducible lac UV5 promoter. This system has been used successfully for the overproduction of many proteins. Alternatively, the polymerase gene can be introduced onto lambda phage by infecting an int-phage such as the commercially available CE6 phage (Novagen, Madison, USA). Other vectors include PLX (Invitrogen, NL) -containing λPL promoters, pTrcHisXpressTm (Invitrogen) or pTrc99 (Pharmacia).
Vector containing the trc promoter (e.g. Biotech, Sweden) or p
Vectors containing a tac promoter such as KK223-3 (Pharmacia Biotech) or PMAL (New England Biolabs, Massachusetts, USA) are included.

Suitable promoter sequences for use with yeast hosts may be regulatable or constitutive, preferably derived from highly expressed yeast genes, especially Saccharomyces cerevisiae genes. Is.
Therefore, TRP1 gene, ADHI or ADHII gene, promoter of acid phosphatase (PH05) gene, a- or promoter of yeast mating pheromone gene encoding α-factor, enolase, glyceraldehyde-3-phosphate dehydrogenase (GAP), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase or gluco Kinase gene, S.
Use a promoter derived from a gene encoding a glycolytic enzyme such as a promoter of the S. cerevisiae GAL4 gene or S. Pombe nmt1 gene, or a promoter derived from the TATA binding protein (TBP) gene be able to.
In addition, the upstream activation sequence (UAS) of one yeast gene and the functional TAT of another yeast gene.
A hybrid promoter comprising a downstream promoter element containing an A box, such as UAS of the yeast PH05 gene and functional TATA of the yeast GAP gene.
Hybrid promoter of a downstream promoter element containing a box
(PH05-GAP hybrid promoter) can be used. Proper configurability PH0
The 5 promoter includes upstream regulatory elements (U, such as the PH05 (-173) promoter element that begins at nucleotide -173 and ends at nucleotide-9 in the PH05 gene.
The truncated acid phosphatase PH05 promoter lacking (AS) is mentioned.

The expression vector is a vector capable of expressing a nucleic acid encoding a polypeptide of interest, is operably linked to a regulatory sequence such as a promoter region, and expresses such DNA. Includes any vector capable of. Thus, expression vectors include plasmids, phages, recombinant viruses, which, when introduced into a suitable host cell, result in the expression of cloned DNA,
Alternatively, it means a recombinant DNA or RNA construct such as other vector. Suitable expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells, and those that are episomal,
Alternatively, those that are integrated into the host cell genome are included.

Recombinant DNA Technique Construction of the vectors of the invention is carried out using conventional ligation techniques. The isolated plasmid or DNA fragment is cut, conditioned, and religated into the desired shape to produce the required plasmid. If desired, analysis is performed by known methods to confirm that the sequences in the constructed plasmid are correct. Construct an expression vector, prepare an in vitro transcript, introduce the DNA into a host cell,
Appropriate methods for performing assays for expression and function are known to those of skill in the art. The presence, amplification and / or expression of a gene in a sample can be determined, for example, by conventional Southern blotting, Northern blotting to quantify mRNA transcription,
It can be directly measured by a dot blotting method (DNA or RNA analysis) or an in situ hybridization method using an appropriate labeled probe based on the sequences provided herein. Those skilled in the art will readily understand how these methods can be modified if desired.

Recombinant Polypeptides Recombinant polypeptides of the invention that may be produced in cells by the methods described herein include, but are not limited to, chymosin, insulin, interferon,
Insulin-like growth factors, antibodies including humanized antibodies, or fragments thereof are included. However, particularly preferred are useful as membrane proteins of prokaryotic and eukaryotic origin, including receptor proteins, chaperone proteins and fragments thereof, proteins of medical and pharmaceutical utility, nucleases and other research tools. Enzymes, proteins used in food processing, including brewing and winemaking, proteins used for detoxification, and proteins used for the decomposition of industrial and household waste.

It has been observed that the polypeptides expressed in the host cells of the invention appear to remain more soluble than the polypeptides that do not. In particular, polypeptides that are only partially soluble in unselected host strains, such as BL21 (DE3), are more soluble or completely lysed when produced in the cells of the invention. To do.

Culture temperature conditions are an important factor in determining the solubility of a polypeptide. Therefore, it is preferable to carry out the culture at a lowered temperature. It is advantageous to culture the cells at between about 30 and about 20 ° C, most preferably at about 25 ° C.

A particularly preferred category of recombinant polypeptides that can be produced by the methods of the present invention are membrane proteins. Conventionally, such proteins have been
Producing it, especially in culture of bacterial cells, has been difficult due to the toxicity of those proteins. Moreover, in conventional expression systems, membrane proteins are not effectively inserted into the membrane during synthesis and are therefore rarely functional. However, the expression system of the present invention provides an effective system for the expression of membrane proteins.

Membrane Composition As described above, a modified membrane composition of a host cell means a membrane composition that differs in some respect from the membrane composition of a wild-type cell. The term "membrane constitution" as used herein means the amount and / or quality of the lipid-containing fraction of cells. It is considered "modified" if its composition differs from that of the wild-type cell. The difference may be a difference in the chemical composition of the lipid fraction, a difference in the mutual proportions of the various lipid-containing components, or other cell fractions, such as protein fractions. Minute, nucleic acid fraction,
Alternatively, it may be the ratio of the lipid fraction to some other cellular fraction. If the membrane composition of the host cell differs in some way from that of the wild-type cell, it is considered "modified".

The modified membrane composition of the host cells of the present invention can be characterized by analytical techniques known to those of skill in the art, which are described below. Preferably, the host cells of the invention have an altered membrane organization, as determined by one or more properties selected from the following: altered lipid to protein ratio, cardiolipin, phosphatidylglycerol. Or, the membrane phospholipid composition is modified with respect to one or more of phosphatidylethanolamines;
Or some other membrane or lipid related property known to those of skill in the art.

The host cell has a lipid to protein ratio of about 0.4 or greater, preferably greater than 0.414, more preferably about 0.5 or greater, even more preferably about 0.6 or greater, even more preferably about 0.7 or greater, or greater. A value is considered to have an altered membrane phospholipid composition.

The host cell contains about 2-4% or more of cardiolipin in its membrane phospholipid composition,
Preferably more than 4.3%, preferably more than 5%, preferably more than 6%, preferably more than 8%, preferably more than 10%, more preferably more than 11%, and further More preferably, it is considered to have an altered membrane phospholipid composition when it is contained in a proportion of more than 11.3% or more.

The host cell will reduce phosphatidylglycerol in its membrane phospholipid composition to about
It is considered to have an altered membrane phospholipid composition when it is included in a proportion of 20% or less, preferably less than 19%, preferably less than 17%, preferably less than 15%, more preferably less than about 13.2%.

A host cell is considered to have an altered membrane phospholipid composition when it contains less than the average level of phosphatidylethanolamine in its membrane phospholipid composition. Average level of phosphatidylethanolamine is about 76.6
% Phosphatidyl ethanolamine. Preferably the host cells of the invention contain less than 76.6% phosphatidylethanolamine, preferably less than 75.45%.

Furthermore, the modified membrane constitution also means that the lipid composition ratio is kept substantially the same, but the morphology of the membrane is modified. In this case, if the morphologically different from the wild-type cells, it is considered that the membrane constitution is modified. A host cell having a modified membrane configuration and a morphological difference as shown herein means that one or more features such as vesicles, inclusion bodies, tube-like structures or networks, or increased lamellae. Being present is included. Other modified membrane morphologies are known to those of skill in the art.

ATP Synthase Subunit As disclosed herein, subunit b or c obtained from the F ₀ membrane region of E. coli ATP synthase (Walker et al., 1982 Nature vol.298, pp867- 869 (
Subunit b); Fillingame (1996) Curr. Op. Struct. Biol. Vol.6 pp491-498
(Subunit c)) is useful for preparing the host cells of the invention. Highly preferred 1
In an embodiment, subunit b of the F ₀ membrane region of E. coli ATP synthase is used in the preparation of host cells of the invention. In yet another embodiment, the invention relates to the use of subunit b or a portion thereof in the preparation of host cells with modified membrane composition of the invention.

Subunit b moieties useful in the present invention include, but are not limited to, subunit b as a whole, subunit b amino acids 1-157, subunit b amino acids 1-
25, amino acids 1-34 of subunit b, amino acids 1-48 of subunit b, amino acids 25-157 of subunit b, or of subunit b that results in a modified membrane organization in a host cell described herein. Any other portion or combination of portions is included. Similarly, portions of subunit c useful in the present invention include, but are not limited to, entire subunit c, or fragments thereof.

Membrane Preparation It is one of the advantageous features of the present invention that the membrane fraction can be easily prepared from host cells as described herein. Conventional preparation of a membrane from cells for expressing a polypeptide requires a high speed centrifugation step of 100,000 × g or more. This step is time consuming and often not only time consuming on fast stages, but also laborious.
Samples should be prepared and infused into single-use tubes, which should then be carefully balanced to the nearest gram, sealed, and centrifuged. Centrifugation itself requires a high-performance vacuum ultracentrifuge, which often requires a large outlay in both initial and operating and maintenance costs. In contrast, the present invention allows membranes to be prepared from host cells without the need for such high speed centrifugation. The modified membranes of the host cells of the invention can be conveniently prepared using only low speed centrifugation at about 2,500-10,000 xg. This method is much cheaper, considerably faster, and substantially more convenient than preparing membranes by conventional methods. This will be discussed in more detail in the Examples section.

The screening polypeptide may be any polypeptide as long as it is desired to identify the interaction with the substance to be screened. However, membrane proteins, especially receptor proteins, are particularly desirable.

Membranes can be obtained from disrupted cells. The cells can be easily isolated, eg by centrifugation, or may be in the form of intact cells. A portion of the membrane fraction from the disrupted cells of the invention is obtained in the form of liposome-like vesicles, which can be immobilized on liposome-specific carriers such as those commercially available from Biacore. .

If desired, the amount of phospholipids in the membrane can be adjusted to approximate the abundance of the polypeptide to be screened in the natural environment.

Methods for investigating ligands that bind to membrane proteins are well known in the art. The main methods used to separate bound and free ligands include rapid filtration, centrifugation, dialysis, gel filtration, sedimentation, or adsorption. Alternatively, the "liposomes" containing the polypeptide to be screened are bound to a carrier compatible with the Biacore system. A library of ligands is prepared and the ligands are screened for their ability to bind to the polypeptide of interest.
Further in v for ligands with high binding constants for the polypeptide of interest
Analyze itro.

Examples Example 1: To produce host cells with modified membrane organization suitable for use in expressing overexpressed polypeptides of E. coli ATP synthase subunits b and c in host cells. The ATP synthase subunits b and c in E. coli host strain C41 (D
It is expressed in E3) and C43 (DE3).

The E. coli host strains C41 (DE3) and C43 (DE3) have already been reported (Miroux
And Walker, 1996).

The unc E and unc F genes encoding subunits c and b of E. coli ATP synthase are amplified by PCR from E. coli DNA. Expression genes pMWI72 (W
ay et al., 1990) and cloned into expression plasmids pMW172 (Ecc) and pMW172 (Ecb).
Are manufactured respectively.

The E. coli cb insert has a forward primer containing a site cleaved by restriction enzymes EcoRI and NdeI: TAG GAA TTC ATA TGG AAA ACC TGA ATA TGG ATC TGC TGT Hind III site reverse primer: CGA AAG CTT TTA TTA CAG It is generated by PCR using TTC AGC GAC AAG TTT ATC CACG and E. coli cells as templates. Replace this PCR product with M
Clone between 13 EcoRI and HindIII sites and confirm by sequencing analysis. Rf M 13 DNA was grown and purified from this clone and the insert was filled with NdeI and H
It is cut out with indIII and ligated into the expression vector PMW172.

The construct for E. coli bc has the following intracistronic sequence: AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA, which is between the DNA sequences for subunits b and c. . This was also made in two separate PCR reactions, one reaction for each subunit, the third reaction was the first
The products from the two reactions are combined. Reaction 1 is for subunit b and is a forward primer that incorporates the following EcoRI and NdeI restriction sites: TAG GAA TTC ATA TGA ATC TTA ACG CAA CAA TCC TCG GCC and a reverse containing the ribosome binding site sequence. Primer: ATC TCC TTC TTA AAG TTA AAC AAA ATT ATT TTA CAG TTC AGC GAC AAG TTT ATC
Is performed using. Reaction 2 is for subunit c and is a forward primer containing the same sequence as the ribosome binding site: TTT GTT TAA CTT TAA GAA GGA GAT ATA ATG GCT TCA GAA AAT ATG ACG CCG and HindIII restriction enzyme cleavage sites. Reverse primer incorporating: CGA AAG CTT TTA CTA CGC GAC AGC GAA CAT CAC GTA CAG. Both of these reactions use E. coli cells as a template. First
The third PCR is performed using the purified product of reaction 1 and the forward primer and the purified product of reaction 2 and the reverse primer. The final product is NdeI and HindIII
Digested with and cloned directly into pMW172. Amino acids 1-25, 1-3 of subunit b
Segments of unc F encoding 4, 1-48, and 26-157 (see examples below) with pM
Cloning into W172 yields plasmids pEc.bN25, pEc.bN34, pEc.bN48, and pEc.b132C, respectively. The vector for co-expression of subunits b and c in pMW172 is made from subunit b promoter proximal (PEc.bc6) and subunit c promoter proximal (pEc.cb4 and pEC.cb5, respectively). Plasmid pEc.cb4 contains the natural intercistronic sequence (Walker et al.,
1984), on the other hand, a ribosome binding site is inserted between genes in pEC.cb5.

Protein Overexpression and Host Cell Harvesting Bacteria were harvested in 2 × TY medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, pH 7.4).
Grow at 37 ° C. in vitro until the absorbance at 600 nm is 0.6. IPTG is then added to a final concentration of 0.7 mM. The cells are further grown at either 37 ° C or 25 ° C and then centrifuged (2,000 xg, 10 minutes). The cells were resuspended in deionized water, re-centrifuged, and 20 volumes of TEP buffer (10 mM Tris, pH8.0, 1 mM EDTA,
And 0.001% (w / v) phenylmethylsulfonyl fluoride). Growth on agar plates is accomplished by preparing plates by adding 1% agar to the liquid medium described above as is known to those skilled in the art.

Culture of E. coli C43 (DE3) grown at 37 ° C entered stationary phase 3-4 hours after induction of subunit b expression, followed by a decrease in cell density (Fig. 1A), while at 25 ° C. The cell density in the stationary phase after expression induction in the culture grown in 1. is kept constant.

The viability of cells obtained from both of the two cultures is examined by plating the cells on TY plates and in the presence and absence of ampicillin and IPTG both alone and in combination. (Miroux and Walker, 1996). Cells from cultures at 37 ° C formed large colonies on all types of plates, whereas colonies from plates containing ampicillin lost the ability to express subunit b, which was 37 ° C. It is shown that the expression of subunit b in E. coli is toxic.

In contrast, samples obtained from cultures at 25 ° C. formed small colonies in the presence of IPTG, and in liquid medium 90% of these colony populations retained the ability to express subunit b. ing.

The kinetics of ATP synthase subunit expression is examined by monitoring the presence or absence of the appropriate protein. Protein concentration is assessed by the bicinchoninic acid assay (Pierce Chemicals, Rockford, IL). Proteins are analyzed by SDS-PAGE in prepared 12-22% gradient gels in Laemmli's buffer (1970). The amino terminal sequence is determined using an Applied Biosystems Procise model 494 protein sequencer. Peptides and proteins are Perkin Elmer
-Investigated by electrospray ionization mass spectrometry on a Sciex API III ⁺ triple quadrupole instrument.

At 25 ° C, initiation of subunit b production is delayed by 2 hours compared to 37 ° C, but the final level of expression of subunit b is almost the same at both growth temperatures (Fig. 1).
(Compare panel B and panel C in).

Subunit c is overexpressed at high levels in both C41 (DE3) and C43 (DE3). Overexpression in C41 (DE3) appears to be toxic at 37 ° C, but less severe than the toxicity associated with subunit b expression. Overgrowth of C41 (DE3) cultures is partially overcome by growing cells at 25 ° C. The culture finally reached an OD ₆₀₀ of 4.7, which contained 2 of subunit c in the culture grown at 37 ° C.
Contains double the amount of subunit c (50 mg per liter of culture). In a pattern similar to that seen for subunit b, overexpression of subunit c in C43 (DE3) at 25 ° C resulted in C
Delayed compared to overexpression in 41 (DE3). In this particular case, the final OD ₆₀₀ of the culture reached about 6.3 and contained a slightly lower amount of recombinant protein (about 30 mg / L culture).

Co-expression of subunits b and c can be achieved by modifying their genes to dicistronic.
ic) Check with placement. Using the gene proximal to the subunit b promoter (plasmid pEc.bc6), very high levels of overproduction of the two proteins are obtained in both mutant hosts. Its growth is similar to cultures overexpressing only subunit b. Using the gene proximal to the subunit c promoter, it is similar to that in the unc operon, and the native intercistronic sequence (Walker et al., 19
84) (plasmid pEc.cb4), the expression of both proteins is low.
Insertion of a synthetic intercistronic sequence (plasmid pEc.cb5) improves expression of both proteins (Fig. 2).

Thus, it has been shown how ATP synthase subunits b and c can be efficiently expressed in host cells. It also shows how their expression can be optimized and enhanced. Furthermore, it is also shown how the toxic effects of such overexpression can be overcome.

Example 2: Induction of Membrane Proliferation in Host Cells It is shown herein that the modified membrane organization of the host cells of the invention can be obtained by expression of subunits of ATP synthase. There is. Expression of the subunit is shown in Example 1 above. As explained above, modified membrane composition refers to the properties of one or more membranes, which properties also include morphological properties.

ATP synthase subunits are expressed in host cells as described in Example 1,
Examine the effect on the morphology of the membrane.

The structure of the film is examined using an electron microscope. Bacteria were first fixed with 2% glutaraldehyde, washed twice with cacodylate buffer (50 mM, pH 7.2), then twice with 4% osmium tetraoxide, then Kellenberger buffer (Kellenberger et al., 1958).
Repeat the washing step 3 times with. The pellet is embedded in 2% agar, cut into 1 mm cubes, and stained with 0.5% (w / v) uranium acetate for 2 hours in the dark. Sample the alcohol (60% ~
Dehydrated with 100%), transferred to propylene oxide and propylene oxide / Epon mixture,
Finally, it is embedded in Epon 812. The resin is polymerized at 60 ° C for 2 days. Thin sections are cut, adsorbed on plastic film coated electron microscopy grids and stained with 2% uranium acetate and lead citrate (Reynolds, 1963).

In another experiment, bacteria are disrupted either by French press or EDTA-lysozyme treatment in combination with osmotic shock (Osborn et al., 1972). The supernatant and pellet were collected by low speed centrifugation (3000 xg) and then adsorbed on a carbon-coated copper grid to give 2%
(w / v) Stain negatively with uranium acetate. Membrane samples obtained from the sucrose density gradient are treated similarly.

All samples were examined in a Philips CM12 transmission electron microscope operating at 120 kV and micrographs are recorded with Kodak film.

Overexpression of subunit b in C41 (DE3) and C43 (DE3) was either taken immediately before induction of expression (FIG. 3, panels E and F) or lacking the expression plasmid (FIG. 3).
, Panels G and H), with an increase in internal structure (FIG. 3, panels AD), not observed in either control.

Although the inner membrane induced in C41 (DE3) is a multi-layered structure, in C43 (DE3) cytoplasmic networks are formed at both 25 ° C and 37 ° C. Under conditions that maximize expression of subunit b, internal structure formation is more extensive in C43 (DE3) than in C41 (DE3) (compare panel A and panel C in Figure 3).

The toxic effect of overexpression of subunit b in C41 (DE3) and C43 (DE3) at 37 ° C. for 18 hours after induction is also evident in the electron micrographs of the bacteria. Large numbers of cells are lysed and the lysis is accompanied by the accumulation of large amounts of extracellular debris. In contrast, at 25 ° C, almost all C43 (DE3) cells overproducing subunit b remained intact (Fig.
3, panel D), remains the same even after forming the internal network. Overproduction of subunit c in both C41 (DE3) and C43 (DE3) is accompanied by an increase in internal multilayer structure similar to that shown in Figure 3 (panels A and B).

Thus, host cells of the invention prepared by expressing subunits of ATP synthase in E. coli strains such as C41 (DE3) and C43 (DE3) were modified as described above. It has been shown that different membrane configurations occur.

Example 3: Preparation of modified membranes Increased membranes obtained from C43 (DE3) overexpressing subunit b shown in Example 2 were grown for 18 hours at 25 ° C after induction. The disrupted cells obtained from the culture are harvested by centrifugation at low speed and collecting the resulting pellet.

Membranes are prepared by harvesting the bacteria and passing the suspension twice through a French press cell at 4 ° C. to disrupt the cells. In some experiments, harvested bacteria were treated with EDTA and lysozyme and subsequently destroyed by osmotic shock (Osb
orn et al., 1972; Yamato et al., 1978).

Escherichia coli C43 (DE3 expressing subunit b, which was grown at 25 ° C. for 18 hours after induction.
) Collect the membranes by centrifuging the cells at 2,500 xg. The same amount of pellets
Resuspend in TEP buffer and centrifuge at 10,000 xg to ensure unbroken cells and cell debris. Otherwise, directly add 2,500 xg of supernatant 10,
Centrifuge at 000 xg. The remaining supernatant is ultracentrifuged at 100,000 xg for 3 hours.

The washed membrane containing subunit b is suspended in TEP buffer at a concentration of 2.5 mg protein / mL. A portion (1 mL) of this suspension is applied to the top of a discontinuous sucrose gradient prepared in a centrifuge tube (14 x 95 mm), but the gradient is 50% (w / v) sucrose cushion ( 2 mL), 30% (w / v) sucrose (3 mL), 10% (w / v) sucrose (3 mL), and 5% (w / v
v) Overlaid with sucrose (2 mL), all 20 mM Tris, pH 8.0, and 0
It was dissolved in a buffer composed of .001% phenylmethylsulfonyl fluoride. Centrifuge the sample at 155,000 xg for 18 hours.

Most of the overexpressed subunit b is in the pellet obtained by low speed centrifugation, and subunit b constitutes about 40% of the protein in that fraction. This level is increased to about 75-80% by the wash step (see Figure 4, lanes b and d). The washed membrane fraction with the highest subunit b content (Figure 4, lane 10) has a buoyant density of 1.18 g / mL on the sucrose density gradient. Smaller fractions with a lower content of subunit b are also found at the position of these gradients, another membrane fraction without subunit b is obtained by ultracentrifugation.

The membrane fraction obtained by low speed centrifugation also contained C43 (DE3 containing the coexpression plasmid pEc.bc6.
) Obtained from cells. Slow centrifugation pellets were not obtained with cells overexpressing only subunit c, and most of the overproduced subunit c was a high-speed centrifugation fraction (100,00
It seems to be at 0 xg) (Fig. 5).

Washed membranes containing subunit b had a tubular or ribbon-like appearance with large vesicles attached (FIG. 6, panel A), a similar structure was obtained by EDTA-lysozyme treatment and osmotic shock. It is also observed in disrupted cells (Osborn et al., 1972) (Fig. 6).
, Panel B). Tubular (or ribbon-like) structures and vesicles have buoyant densities of sucrose gradients of 1.10 g / mL and 1.18 g / mL, respectively. Vesicles contain more subunit b than the ribbon-like structure (Figure 6, panels C and D). After EDTA-lysozyme treatment, cells did not swell, indicating that the intracytoplasmic network is not an extension of the E. coli inner membrane.

Thus, the present invention provides a convenient method for the preparation of modified membranes of host cells, as described herein. This method is particularly advantageous because it requires only low speed (2,500-10,000 xg) centrifugation and no need for expensive and time consuming high speed centrifugation.
In addition, the morphological characteristics of the modified membranes of the host cells of the invention are shown here.

Example 4 Membrane Characterization of Host Cells Inner Membrane Composition Under the majority of growth conditions, bacteria maintain a constant lipid: protein ratio (Cronan and
Rock, 1984), and 0.4 is a typical value for the inner membrane of E. coli (Ingraham et al., 1983).

In C41 (DE3) and C43 (DE3) cells that do not overexpress subunit b, the ratio is normal (see Table 1), while C43 overexpressing subunit b is present. (DE3)
In cells, the values of lipid content and ratio for increased membranes are much higher. This high lipid content reflects the high viscosity of the membrane. In contrast, the values reported for membranes of E. coli cells overexpressing fumarate reductase, which can be accompanied by an increase in internal membranes, are close to normal (Weiner et al., (1984)
.

[0103]

[Table 1] Table 1. Increase with overexpression of ATP synthase subunit b in Escherichia coli C43 (DE3)
Phospholipid content of the permeated membrane The above values were obtained from 3-4 independent measurements.

To further characterize the modified membranes of the host cells of the invention, membrane phospholipid analysis is performed.

Lipids are extracted according to the method of Bligh and Dyer (1959). 1 for part of the membrane fraction
M HCl (20 μL) is added with water to bring the total volume to 200 μL. Chloroform: methanol (volume ratio 1: 2.2, 640 μL) is added and the sample is vortexed for 1 minute. Then 0.1 M HCl (200 μL) and chloroform (200 μL) are added and the sample is vortexed again for 1 minute. Centrifuge the mixture and transfer the chloroform phase. Chloroform (200
μL) is added and mixed with the aqueous phase for 1 minute, then the mixture is centrifuged. The chloroform fractions were pooled and buffered (50 mM Tris-HCl, pH8.25, 0.1M NaCl, 0.1M EGTA.
). The mixture is centrifuged, the chloroform phase is transferred and evaporated. The lipid extract is hydrolyzed with perchloric acid (300 μL) at 180 ° C. for 3 hours and then the phosphorus concentration is measured (Rouser et al., 1970).

The phospholipid composition (150 nmoles of phosphorus) of the lipid extract of the membrane fraction was subjected to thin layer chromatography on silica 60 plate using 65:25:10 (volume ratio) of chloroform: methanol: acetic acid as a solvent. Analyze by TLC). Visualize lipid with iodine vapor, scrape,
Boil and centrifuge to remove traces of silica and measure phosphorus content as described above.

Phosphatidic acid (PA) and cardiolipin (CL) do not dissolve in this system,
These are soluble in the other two systems; the first system is a two-dimensional system and chloroform: methanol: water: ammonia (volume ratio 68: 28: 2: 2) is the first dimension solvent, chloroform: methanol. : Acetic acid (volume ratio 65:25:10) is used as the second-dimensional solvent.

In the second system (Fine and Sprecher, 1982), the phospholipids were chloroform: methanol: water: ammonia (volume ratio 120: 75: 120: 1) on silica plates pretreated with 1.2% boric acid.
6: 2) is separated as a solvent. No phosphatidic acid is found in the sample using either chromatographic system.

Phospholipids on silica plates are visualized by spraying with phosphoreagent (molybdenum (IV) oxide), then heated until a blue color appears.

The average phospholipid composition of E. coli cells is 76% phosphatidylethanolamine, 20% phosphatidylglycerol, with small amounts of cardiolipin and unidentifiable species (Ames, 1968; Raetz et al., 1978; Randle et al. , 1969; Cronan, 1968).
The composition of the main group is unaffected by changing growth conditions, but is changed during the growth cycle and phosphatidylglycerol is partially converted to cardiolipin by condensation of two molecules of phosphatidylglycerol in the early stationary phase. Be done (Cronan and Vagelo
s, 1972). It has been suggested that cardiolipin may help bacterial survival, perhaps by ensuring a minimal membrane phospholipid content and stabilizing membrane structure when the bacteria are not actively growing and dividing. (Hiraoka et al., 19
93).

Membranes isolated from C41 (DE3) and C43 (DE3) cells in which subunit b is not expressed contain about 2-4% cardiolipin but overexpress subunit b. In C43 (DE3), increased membrane cardiolipin levels showed a dramatic increase to about 14% by consuming its biosynthetic precursor phosphatidylglycerol, with phosphatidylglycerol at about 20% in controls. % To about 13% (see Table 1)
). The level of phosphatidylethanolamine in the increased membrane is also slightly lower than the control.

Cardiolipin has been proposed to play a role in transporting proteins across the membrane, and anionic phospholipids modulate membrane insertion (Liu et al., 19
97; van Klompenburg et al., 1997) and have been shown to act as chaperones in the assembly of membrane proteins (Bogdanov et al., 1996). Thus, the observations reported herein indicate that the host strains of the invention are useful in facilitating the overexpression, folding, and membrane insertion processes of membrane proteins.

The increase in cardiolipin content with overexpression of subunit b does not appear to be the result of mutations in the cardiolipin biosynthetic pathway, as normal levels in control cells. In C41 (DE3) and C43 (DE3), the onset of protein overproduction is BL21 (DE3
), Not only improves the coupling between transcription and translation, but also improves the coupling between subunit b production and cardiolipin biosynthesis.

Subunit b is incorporated into the internal membrane. Subunit b of Escherichia coli ATP synthase has a transmembrane N-terminal hydrophobic segment,
It is followed by a highly charged hydrophilic domain that projects from the membrane and interacts with the F ₁ catalytic domain (Walker et al., 1982). In isolated F ₀ , the superficial domain of subunit b is proteolytically, thereby preventing F ₁ reassociation (Perlin et al., 1983).

Therefore, in order to investigate whether the overexpressed subunit b was correctly incorporated into the increased membrane of the host cell of the present invention, the distribution state of the subunit b in the isolated membrane was examined by trypsin degradation. .

Membranes are isolated from elevated E. coli C43 (DE3) at 25 ° C. that overproduced subunit b (see above) and are suspended in TEP buffer at a concentration of 2.5 mg protein / mL. Trypsin (1:20, w / v) is added and the suspension is kept at 30 ° C for 1 hour. Samples are taken at regular time intervals and soybean trypsin inhibitor (5-fold excess by weight) is added to terminate protein degradation. The progress of proteolysis is monitored by SDS-PAGE analysis of the fragment polypeptide. Sample cooled to 4 ° C and membrane measured at 128,000 xg
C1 Aquapore RP-300 column (particle size 7 μm, pore size 300 Å, 100 mm x 2.1 mm ID; Applied Bi
Peptides are isolated by reverse phase chromatography using osystems, Warrington, Cheshire, UK). Equilibrate the column with 0.1% trifluoroacetic acid and elute with a linear density gradient of acetonitrile. Peptides were analyzed by electrospray ionization mass spectrometry, separated by SDS-PAGE, transferred to poly (vinylidene difluoride) membrane,
Stain with PAGE Blue 83 dye and analyze by N-terminal sequence analysis.

[0117] After digestion for 5 minutes, M _r values of apparent 12,8,7, and major proteolytic fragments of four is 6 kDa is detected in the soluble fraction (Figure 7A). They all have SQILDEKAE ... as the N-terminal sequence, which corresponds to residue 83 and beyond. Analysis of peptides in pellet fractions by SDS-PAGE (Figure 7B) revealed
M _r indicates the fragment is a 7 kDa. This fragment was only slightly stained with Coomassie blue dye, indicating that the fragment is hydrophobic. That
The N-terminus is the same as intact subunit b, and the molecular weight measured by electrospray ionization mass spectrometry is 4113.3, which is 1-36 of subunit b.
It corresponds to the second residue (calculated molecular weight 4115.1) and results from cleavage by trypsin after arginine-36. There is no evidence of cleavage behind lysine-23, which is suggested by the NMR-examined structure, which suggests that the region around this residue is in an α-helix associated with the membrane, which causes trypsin. This is consistent with being protected from degradation (Dimitriev et al., 1999).

These features are consistent with subunit b being correctly inserted into the membrane, indicating that the host cells of the invention provide the correct folding and processing of the polypeptide expressed in that cell. There is.

Example 5: Production of different membrane features by expression of different fragments of the ATPase subunit. To investigate the conditions necessary to bring about an increase in intracellular membrane structure in E. coli, the intact protein is examined. The truncated form of subunit b is introduced into C41 (DE3) and C43 (DE3) with an expression vector under the same conditions used for.

The fragments used in this example correspond to amino acids 1-25, 1-34, 1-48, and 25-157 of subunit b. Peptides 1-25 and 1-34 are hydrophobic and form the transmembrane portion of subunit b. Inclusion bodies are observed in electron micrographs of cell sections when peptides 1-25 or 1-34 are expressed in C41 (DE3), and intracellular membrane structures are not observed when expressed in C43 (DE3). .

Expression of peptide 1-48 induces the formation of vesicular structures in C41 (DE3),
The formation of inclusion bodies is induced in DE3). Residues 25-157, which lack the hydrophobic domain of subunit b, but whose expression is lower than that of the intact protein in both C41 (DE3) and C43 (DE3) cells The morphology is different with the increase of the membrane (see Table 2).

[0122]

[Table 2] This indicates the utility of expressing the subunit b peptide of the present invention or a fragment thereof for the purpose of producing a host cell having an altered membrane composition such as an altered membrane morphology.

Example 6 Expression of Polypeptides in Host Cells of the Invention Rhodobacter sp. Is a heterologous overproduction of membrane proteins associated with intracellular membrane increase.
It has been observed in bacterial light-harvesting complexes in heroides (Fowler et al., 1995), Saccharo.
PMA2A in myces cerevisiae (Supply et al., 1993) and others (Ohkuma et al., 1995)
It has been observed that overexpression of TPase is accompanied by an increase in intracellular membranes. It has been suggested that some of these systems are general tools for overexpressing recombinant integral membrane proteins (Fowler et al., 1995; Gong et al., 1996). However, the genetics of E. coli are well known, making it an ideal host for overproduction of membrane proteins.

Bacterial overexpression of membrane proteins can be improved by optimization of the host system. To overproduce the membrane at high levels, transcription and translation must be coordinated, as are phospholipids and membrane protein synthesis, folding, and insertion.

In the case of overproduction of subunit b in C43 (DE3), the incorporation of the protein in its membrane-soluble form leads to the formation of a network of tubular structures. Both the phospholipid composition and the structure of the recombinant protein are considered to influence the formation of these structures.

A similar effect is associated with co-expression of subunits b and c when the gene for subunit b is proximal to the promoter, but not proximally.

Thus, by overexpressing membrane proteins and expressing them in tandem with subunit b in the host cells of the invention, they arrange the folding and insertion of the membrane proteins into the membrane. It is possible.

[0128]References Ames, G.F. (1968) Lipids of Sa. Salmonella typhimurium and E. coli; Structure and metabolism
lmonella typhimurium and Escherichia coli: structure and metabolism J.
Bacteriol. 95, 833-843. Bogdanov, M., Sun, J., Kaback H.R. & Dowhan W. (1996). Membrane transport protein
Phospholipids act as chaperones in the assembly of (A phospholipi
d acts as chaperone in assembly of a membrane transport protein.) J. Bio
l. Chem. 20, 1161511618. Bligh, E.G. & Dyer, W.J. (1959). A rapid method for extraction and purification of total lipids (A rapid
 method of total lipid extraction and purification) Can. J. Biochem. Phy
siol. 37, 911. Cronan, J. E. (1968). Changes in phospholipids during the increase of Escherichia coli (Phospholipid al.
teration during growth of Escherichia coli.) J. Bacteriol. 95, 2054-2061 Cronan, J. E. & Vagelos, P.R. (1972). Metabolism and function of membrane phospholipids in Escherichia coli (M
etabolism and function of the membrane phospholipids of Escherichia coli
.) Biochim. Biophys. Acta 265, 25-60. Cronan, J. E. & Rock C.O. (1984). Biosynthesis of Membrane Lipids
ane lipids.) Description in Escherichia coli and Salmonella typhimurium, Nedhar
t, F.C. (ed.) American Society for Microbiology, Washington D.C. De Kruijff, B. (1987) News and Views. Nature 329, 587-588. Dimitriev, O., Jones, P.C., Jiang, W. & Fillingame, R.H. (1999). E. coli F
₀F₁ Structure of the membrane domain of subunit b of ATP synthase (Structure of the me
mbrane domain of subunit b of the Escherichia coli F₀F₁ (ATP synthase.)
 J. Biol. Chem. 274, 15598-15604. Dong, H., Nilsson, L. & Kurland, C.G. (1995). Unknown cause of gene in E. coli.
Overexpression leads to increased inhibition and ribosome destruction (Gratuitous over
expression of genes in Escherichia coli leads to growth inhibition and r
ibosome destruction.) J. Bacteriol. 177, 1497-1504. Fillingame, R.H. (1996). F- and V-Type H⁺Membrane sector of transportable ATPase (Me
mbrane sectors of F_- and V-type H₊-tranporting ATPase.) Curr. Op. Struc
t. Biol. 6: 491-498. Fine, J. B. & Sprecher, H. (1982). Reprocessing on plates impregnated with boric acid.
Unidimensional thin-layer chromatograph
graphy of phospholipids on boric acid impregnated plates.) J. Lipid Res.
23: 660-663. Fowler, G.J.S., Gardiner, A.T., MacKenzie, R.C., Barrat, S.J., Simmons,
A.E., Westerhuis, W.J., Codgell, R.J. & Hunter, C.N. (1995). Rhodobacter
Heterologous expression of the gene encoding the bacterial light-harvesting complex in spaeroides (Heter
ologous expression of genes encoding bacterial light harvesting complexe
s in Rhodobacter sphaeroides.) J. Biol. Chem. 270: 23875-23882. Girvin, M.E., Rastogi, V.K., Abildgaard, F., Markley, J.L. & Fillingame,
 R.H. (1998). F₁F₀ Transmembrane H of ATP synthase⁺Solution structure of transport subunit c (S
olution structure of the tansmembrane H⁺-transporting subunit c of the F ₁ F₀ ATP synthase.) Biochemistry 37, 8817-8824. Glazebrook, T.S., Cannon, G.C. & Shiveley, J.M. (1979). In E. coli 0111a
Of intracellular cytoplasmic membrane structure: cell membrane isolation and lipid characterization (Intracytoplasmic m
embrane production in Escherichia coli 0111a: Isolation and lipid charac
terization of cell membranes.) Current microbiology 2: 5-10. Gong, F.C., Giddings, T.H., Meel, J.B., Staehelin, L.A. & Galbraith, D.W
. (1996). Z Membrane: Intracellular artificial device for overexpression of recombinant integral membrane protein
Official (Z-membranes: artificial organelles for overexpressing recombinant in
Integral membrane proteins.) Proc. Natl. Acad. Sci. USA. 93: 2219-2223. Hiraoka, S., Matsuzaki, H. & Shibuya, I. (1993).
Active increase in cardiolipin synthesis and its physiological significance
 cardiolipin synthesis in the stationary growth phase and its physiologi
cal significance in Escherichia coli.) FEBS Letters 336: 221-224. Hockney, R.C. (1994). Recent developments in heterologous protein production in E. coli (Recent
 developments in heterologous protein production in Escllerichia coli.)
Trends Biotechnol. 12: 456-463. Ingraham, J.L., Maaloe, O. & Neidhart, F.C. (1983). Increase in bacterial cells (Grow
th of the bacterial cell.) Sinauer Associates. Sunderland. Mass. Kellenberger, E. & Ryter, A. (1958). Escherichia coli cell wall and cytoplasmic membrane structure.
Structure (Cell wall and cytoplasmic membrane of E. coli.) J. Biophys Biochem.
Cytol. 4: 323-326. Kurland, C.G. & Dong, H. (1996). Preventing bacterial increase due to protein overproduction.
Harm (Bacterial growth inhibition by overproduction of protein.) Molec. Mi
crobiol. 21: 1-4. Laemli, U.K. (1970). Structure of T4 bacteriophage head assembly.
Cleavage of structural proteins during the assembly of
 the head of bacteriophage T4.) Nature 227: 680-685. Liu, L.P., & Deber, C.M. (1997). Anionic phospholipids insert peptides into membranes.
Anionic phospholipids modulate peptide insertion into memb
ranes.) Biochemistry 36: 5476-5482. Miroux, B. & Walker, J.E. (1996). Overproduction of proteins in E. coli: how many
Escherichia coli enables high-level synthesis of various membrane and globular proteins
Mutant host (Over-production of proteins in Escherichia coli: mutant host
s that allow synthesis of some membrane proteins and globular proteins a
t high levels.) J. Mol. Biol. 260: 289-298. Ohkuma, M., Park, S.M., Zimmer, T., Menzel, R., Vogel, F., Schunck, W. H
., Ohta, A. & Takagi, M. (1995). Cytochrome P-450 in Candida maltosa
Proliferation of intracellular membrane structure during homologous overproduction
 membrane structures upon homologous overproduction of cytochrome P-450
in Candida maltosa.) Biochim. Biophys. Acta. 1236: 163-169. Osbom, M.J., Gander, J.E., Parise, E. & Carson, J. (1972).
Mechanism of fungal adventitia assembly. Isolation and characterization of cytoplasmic and outer membranes (Me
chanism of assembly of the outer membrane of Salmonella Typhimurium. Iso
lation and characterization of cytoplasmic and outer membrane.) J. Biol. C
hem. 247: 3962-3972. Perlin, D.S., Cox, D.N. & Senior, A.E. (1983). Proton-ATPase of Escherichia coli
F₁And incorporation of the membrane sector. Role of subunit b (unc F protein) (I
ntegration of F₁ and the membrane sector of the proton-ATPase of Escheri
chia coli. Role of subunit b (uncF protein).) J. Biol. Chem. 258; 9793-9800
. Raetz, C.R.H. (1978). Enzymes, genetics, and regulation of membrane phospholipid synthesis in Escherichia coli.
Section (Enzymology, genetics, and regulation of membrane phospholipids synth
esis in Esclierichia coli.) Microbiol. Rev. 42: 614-659. Randle, C.L., Albro, P.N. & Dittmer, J.C. (1969). Phosphorus of Gram-negative bacteria
The composition of glycerides and their composition when increasing (The phosphoglyceride composi
tion of Gram-negative bacteria and the chances in composition during gro
wth.) Biochim. Blophys. Acta 187: 214-220. Rouser, G., Fleischer, S. & Yamamoto, A. (1970). Two-dimensional thin layer of polar lipids.
Determination of phospholipids by chromatographic separation and spot phosphorus analysis (
Two dimensional thin-layer chromatographic separation of polar lipids an
d determination of phospholipids by phosphorus analysis of spots.) Lipid
s 5: 494-496. Schnaitman, C. & Greenwalt, J.W. (1966). Intrac membrane structure of Escherichia coli (Intrac
ytoplasmic membranes in Escherichia coli.) J. Bacteriol. 92: 780-783. Studier, F.W., Rossenberg, A.H., Dunn, J.J. & Dubendorff, J.W. (1990).
Use of T7 RNA polymerase to direct expression of cloned genes (Use of
 T7 RNA polymerase to direct expression of cloned genes.) Methods Enzymo
l. 185: 60-89. Supply, P., Wach, A., Thines-Sempoux, D., & Goffeau A. (1993). Saccharom
Increased intracellular structure of PMA2 ATPase in yces cerevisie (Prol
iferation of intracellular- structures upon overexpression of the PMA2 A
TPase in Saccharomyces cerevisie.) J. Biol. Chem., 268: 19744-19752. van Klompenburg, W., Nilsson, I., von Heijne, G., & de Kruijff, B. (1997
). Anionic phospholipids are determinants of protein morphology. (Anionic phosp
holipids are determinant of protein topology.) EMBO J. 16: 4261-4266. von Meyenburg, K., Jorgensen, B.B. & Reurs, B. (1984). E. coli K-12 strain
And morphological effects of overproduction of membrane-bound ATP synthase in culture
(Physiological and morphological effect of overproduction of membrane-bo
und ATP synthase in Escherichia coli K-12.) EMBO J., 3: 1791-1797. Walker, J.E., Saraste, M., & Gay, N.J. (1982). E. coli F₁-ATP synthase
Interacts with the protein component of the proton channel (E. coli F₁-
ATP synthase interacts with a membrane protein component of a proton cha
nnel.) Nature, 298: 867-869. Walker, J.E., Saraste, M., & Gay, N.J. (1984). Unc operon: ATP synthesis
Nucleotide sequence control and structure of the enzyme (The unc operon.
gulation and structure of ATP synthase.) Biochim. Biophys. Acta, 768: 164
-200. Walker, J.E. & Saraste, M. (1996) Membrane protein structures
tructure.) Editorial overview. Curr. Op. Struct. Biol., 6: 457-459. Weigand, R.A., Shiveley, J.M. & Greenwalt, J.W. (1970). Escherichia coli outer membrane shape
Formation and ultrastructure of extramembranes in Escheric
hia coli.) J. Bacteriol., 102: 240-249. Weiner, J.H., Lemire, B.D., Elmes, M.L., Bradley, R.D. & Scraba, D.G. (1
984) Overproduction of fumarate reductase in Escherichia coli is a novel intracellular lipid-protein
Induction of organelles (Overproduction of fumarate reductase in Escherichia co
li induces a novel intracellular lipid-protein organelle.) J. Bacteriol.,
 158: 590-596. Wilkinson, W.O., Walsh, J.M., Corless, J.M. & Bell, R.M. (1986).
Escherichia coli sn-glycerol-3-phosphate acyl transfer
Crystalline arrays of the Escherichia coli sn-glycerol
-3-phosphate acyltransferase, an integral membrane protein.) J. Biol. Chem
.261: 9951-9958. Yamato, I., Futai, M., Anraku, Y. & Nonomura, Y. (1978). E. coli cytoplasm
Cytoplasmic membrane vesicles of Escherichia coli. J. Biochem.
, 83: 117-128.

[Sequence list]

[Brief description of drawings]

FIG. 1 shows the effect of expression of E. coli F-ATPase subunit b on the growth of E. coli C43 (DE3), graph (panel A), and Coomassie-stained polypeptide (
Panels B and C). Fresh colonies of host cells containing plasmids for overexpressing subunit b are inoculated into 2xTY medium supplemented with ampicillin. The culture is grown at 37 ° C. for 4-5 hours and IPTG (0.7 mM) is added when the OD at 600 nm of the culture reaches 0.6. The cells are then grown at either 37 ° C or 25 ° C. Panel A: Growth of C43 (DE3) at 25 ° C (○) and 37 ° C (●). Panels B and C: Sample 10 of C43 (DE3) culture grown at 37 ° C and 25 ° C, respectively.
Analysis by μL SDS-PAGE. Subunit b is a band of about 20 kDa.

FIG. 2 shows a Coomassie-stained polypeptide showing co-expression of E. coli C43 (DE3) E. coli F-ATPase subunits b and c. Expressions from plasmids pEc.bc6 (bc) and pEe.cb5 (cb) are compared. Freshly transformed colonies of C43 (DE3) are inoculated into 2xTY medium (50 mL) supplemented with ampicillin. Cultures at 37 ° C 4
Grow for ~ 5 hours and add IPTG (0.7 mM) when the OD at 600 nm of the culture reaches 0.6. The cells are then grown at 37 ° C for 1, 2 or 3 hours. 20 μL of culture is analyzed by SDS-PAGE at the times indicated on each slot. E. coli F ₁ F ₀ -ATPase (ATP synthase) (1 μg) is loaded as a marker in the center lane. The positions of their various subunits are shown on the right.

FIG. 3 shows an electron micrograph of a thin section of Escherichia coli cells overproducing subunit b of Escherichia coli ATP synthase. Panels A and B: 37 ° C after induction of expression
Or C41 (DE3) cells overproducing subunit b, grown at 25 ° C. for 3 hours and 18 hours, respectively. Panels C and D: Subunit b overproducing C43 (DE3) cells grown for 3 and 18 hours at 37 ° C or 25 ° C, respectively, after induction. Panels E and F: C41 (DE3) and C43 (DE3) cells containing the plasmid without insert grown at 37 ° C. for 4 hours after adding IPTG to the culture. Panels G and H: Plasmid-free C41 (DE3) and C43 grown under the same conditions as above
(DE3) cells. The scale bar shows 0.2 μm in panels A to D and 0.28 μm in panels E to H.

FIG. 4 shows a Coomassie-stained polypeptide showing the protein content of increased membranes isolated from E. coli C43 (DE3) cells overproducing subunit b. After induction of expression, cells are kept at 25 ° C for 18 hours and samples are analyzed by SDS-PAGE. Lane (a): whole cell extract (10 μL); lane (b): pellet by low speed centrifugation (3,000 × g, protein 30 μg); lane (c): supernatant obtained from lane (b) by high speed centrifugation ( Membrane fraction (35 μg protein) obtained by 100,000 × g); Lane (d): High-speed pellet (20 μg protein) obtained by resuspending and washing the substance in lane (b). Lane (1-11): Fraction obtained from sucrose step gradient fractionation of the material in lane (d) (5 μL sample from 1 mL fraction).

FIG. 5: E. coli C overexpressing subunits b and c of E. coli ATP synthase
4 shows a Coomassie-stained polypeptide showing the protein content of modified membranes isolated from 43 (DE3) cells. Samples are analyzed by SDS-PAGE. Lane (a) and (
b) includes only subunit b and subunits b and c together (bc
(Disposition genes), pellets obtained by low-speed centrifugation (3,000 xg) from overproducing cells (
Protein 25 μg) is shown. Lane (c) shows a pellet (35 μg of protein) obtained by high speed centrifugation (100,000 × g) from cells overexpressing only subunit c. The intensity band with an apparent molecular weight of 16 kDa is identified as the heat shock protein Hsp16 by protein sequencing.

FIG. 6 shows an electron micrograph of negative staining of isolated increasing membranes. The membrane was constructed in Escherichia coli C43 (DE3) host cells overexpressing subunit b by French press (
Panel A), or disrupted by osmotic shock (panel B) after EDTA and lysozyme treatment, and then obtained by low speed centrifugation (3,000 × g). The former membrane is fractionated by sucrose density gradient, negatively stained and analyzed by electron microscopy. Panel C shows fraction 10 of the sucrose density gradient (see Figure 3, buoyant density 1.18 g / mL). Panel D shows fraction 7 of the sucrose density gradient (see Figure 3, buoyant density 1.10 g / mL). (Scale bar 0.28 μm)
.

FIG. 7 shows a Coomassie-stained polypeptide obtained as a result of trypsin degradation of subunit b of Escherichia coli ATP synthase incorporated into a modified membrane. Supernatants (panel A) and pellets (panel B) are each obtained by trypsin digestion for the times indicated and analyzed by SDS-PAGE. The M _{r 12} , 8, 7 and 6 kDa bands observed in the supernatant after 5 minutes of digestion (indicated by the arrow on the left in panel A) show the same N-terminal sequence up to residue 83. Have
In panel B, the band with an M _r of 7 kDa indicated by the arrow corresponds to residues 1-36 of subunit b.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Milox, Bruno, Donat, Mike             Le             France F-92190 Medon, Ryu               Gee. Etzel, 9, CNR             S-Celemod (72) Inventor Arechaga, Ignacio             UK CB2             2 Xwai, Hills Road, Well             Come Trust / MRC Bilde             Ding, MRC Dan Huma             Nutrition unit F term (reference) 4B024 AA01 BA80 CA04 DA06 EA04                       FA02 GA11 HA01 HA03                 4B063 QA01 QA18 QQ06 QQ13 QQ79                       QR33 QR59 QR75 QR80 QS05                       QS12 QS36 QX02                 4B065 AA26X AA26Y AB01 BA01                       CA24 CA44

Claims (26)

[Claims] 1. A method for producing a polypeptide, which comprises the following steps: a) a bacterial host cell comprising a cytoplasmic membrane, the intracytoplasmic membrane being other than the host cell. A host cell having a configuration different from that of the membrane of b); b) transforming the host cell with a nucleic acid construct containing a coding sequence encoding the polypeptide, In a sex host cell so that its expression is associated with the cytoplasmic membrane; and c) isolating the polypeptide by separating the cytoplasmic membrane from other cellular constituents. A method comprising: 2. The method of claim 1, wherein the production of intracellular membranes is induced in the bacterial host cell prior to or concomitant with expression of the polypeptide. 3. The characteristics of the cytoplasmic membrane consisting of other membranes in host cells and the following groups: a) a lipid: protein ratio of greater than about 0.4; b) a phospholipid composition of about 4% or less. Including large cardiolipin; c) having a phospholipid composition of less than about 20% phosphatidylglycerol; and d) having a lower than normal level of phosphatidylethanolamine in the membrane. 3. The method of claim 1 or claim 2 differing in that it has one or more properties selected from the group consisting of: 4. The production of a cytoplasmic membrane is induced by the expression in the host cell of subunits b and / or c of ATP synthase capable of inducing an increase in the membrane, or a fragment thereof. The method of claim 2 or claim 3, wherein: 5.   The method according to claim 4, wherein the ATP synthase is the F0 membrane region of Escherichia coli ATP synthase. 6. The ATP synthase fragment is amino acids 1-25, 1-34, 1-2 of subunit A of ATP synthase.
6. The method of claim 4 or claim 5 consisting essentially of 8, 1-157, or 25-157. 7. A host cell comprising a coding sequence encoding subunits b and / or c of ATP synthase or a fragment thereof, which induces an increase in the membrane and is operably linked to a promoter. Which is transformed with the nucleic acid construct of
The method according to any one of 4 to 6. 8. The method according to claim 7, wherein the subunits b and / or c of ATP synthase or a fragment thereof capable of inducing increase of membrane and the polypeptide are encoded on the same nucleic acid construct. Method. 9. A coding sequence encoding subunits b and / or c of ATP synthase or a fragment thereof capable of inducing an increase in the membrane is placed upstream of the coding sequence encoding the polypeptide, 9. The method of claim 8, wherein the two coding sequences are expressed in tandem from the same promoter. 10.   The method according to any one of claims 1 to 9, wherein the host cell is an E. coli cell. 11. The cells are Escherichia coli C43 (DE3) (ECCC B96070455) and Escherichia coli C41 (DE3) (ECCC B96070444).
11. The method according to claim 10, which is the cell itself selected from the group consisting of E. coli DK8 (DE3) S (NCIMB 40885) or Escherichia coli C2014 (DE3) (NCIMB 40884), or derived therefrom. 12. The method according to any one of claims 1 to 11, wherein the polypeptide is a membrane protein. 13. Polypeptide and / or subunits b and / or c of ATP synthase
13. The method according to any one of claims 1-12, wherein the coding sequence coding for is operably linked to an inducible promoter. 14. A bacterial host comprising a coding sequence coding for said polypeptide and a coding sequence coding for the b and / or c subunit of ATP synthase or a fragment thereof capable of inducing an increase in membranes. A nucleic acid construct for expression of a polypeptide in. 15. A coding sequence coding for the b and / or c subunit of ATP synthase or a fragment thereof capable of inducing an increase in the membrane is placed upstream of the coding sequence coding for said polypeptide. 15. The nucleic acid construct according to claim 14, which is present. 16. A coding sequence encoding a b and / or c subunit of ATP synthase or a fragment thereof capable of inducing an increase in membrane, and / or a coding sequence coding for said polypeptide is inducible. The nucleic acid construct according to claim 14 or 15, which is under the control of a promoter. 17. A coding sequence encoding a b and / or c subunit of ATP synthase or a fragment thereof capable of inducing an increase in membrane, and a coding sequence coding for the polypeptide are of the same promoter. The nucleic acid construct according to claim 16, which is under control. 18. The nucleic acid construct according to any one of claims 14 to 17, further comprising a coding sequence encoding a detectable label. 19.   19. The nucleic acid construct according to claim 18, wherein the detectable label is green fluorescent protein. 20. The nucleic acid construct according to any one of claims 14 to 19, wherein at least one of the coding sequences is under the control of a promoter recognized by bacteriophage RNA polymerase. 21. The nucleic acid construct according to any one of claims 14 to 20, wherein the ATP synthase is the F0 membrane region of Escherichia coli ATP synthase. 22. ATP synthase fragments are amino acids 1-25, 1-34, 1-4 of subunit b of ATP synthase.
22. A nucleic acid construct according to any one of claims 14 to 21 consisting essentially of 8, 1-157, or 25-157. 23.   A host cell comprising a nucleic acid construct according to any one of claims 14 to 33. 24. The host cell according to claim 23, which is the Escherichia coli cell according to claim 10 or 11. 25. A method of screening for an agent that binds to, affects, or modifies a desired membrane protein, the method comprising:
14. A host cell is transformed with the vector according to any one of 14 to 24 to induce expression of a desired membrane protein, and the host cell is cultured to produce the desired membrane protein. Immobilized on a carrier, and exposing the membrane to the drug to be screened under conditions that promote the drug-polypeptide interaction. 26. A method of screening for an agent that binds to, affects, or modifies a desired polypeptide, the method comprising the steps of: a) claims 14-24. Transforming a host cell with the vector according to any one of the items; b) the subunit b or subunit c of the ATP synthase from the first expression unit
And culturing the host cell so that the production of the membrane is induced; c) inducing expression of the desired membrane protein from the second expression unit and culturing the host cell to produce the desired Producing a membrane protein; and d) immobilizing the cells on a carrier and exposing the cells to the agent to be screened under conditions that promote the interaction of the agent with the polypeptide.