CA2387180A1 - Expression system for membrane proteins - Google Patents

Abstract

The invention relates to a method for preparing a polypeptide comprising: expressing said polypeptide in a host cell for use in protein expression, wherein said host cell has an altered membrane composition, isolating the membrane fraction from said host cell, and isolating said polypeptide from said membrane fraction.

Description

EXPRESSION SYSTEM FOR MEMBRANE PROTEINS
Field of the Invention The invention relates to an expression system for polypeptides. In particular, the invention relates to host cells for polypeptide expression which have an altered membrane composition.
Background to the Invention The over-expression of many proteins in Eschenichia coli impairs the growrth of the bacteria, or even kills them. These toxic effects are particularly severe with membrane proteins. One factor impeding the analysis of membrane proteins is the lack of generally applicable systems for their over-expression (Walker and Saraste, 1996).
1 ~ Although E. coli is often a successful vehicle for over-expression of both prokaryotic and eukaryotic proteins (see Hockney, 1994), the expression of most membrane proteins and of many globular proteins kills the host bacteria (Dong et al., 1995;
Kurland and Dong, 1996; Miroux and Walker, 1996).
This problem applies particularly to the widely used bacterial expression system in which the target gene is transcribed by bacteriophage T? RNA polymerase in the host strain E. coli BL21 (DE3) (Studier et al., 1990).
In order to try and overcome these difficulties for the T7 system, mutant hosts were 2~ selected from E. coli BL21(DE3) that allow over-production of some membrane proteins and of some globular proteins which could not be expressed in BL21(DE3) without exhibiting toxic effects. These mutant host strains are E. coli C41 (DE3) and C43(DE3). In some cases, where expression levels were low in BL21(DE3), slightly better expression was obtained in C41 (DE3) or C43(DE3) (Miroux and Walker, 1996).

However, overexpression of many polypeptides is still problematic, even using these C41/C43 strains.
The present invention seeks to overcome problems associated with prior art 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 according to the invention have and altered membrane composition in comparison with other cell membranes, which is useful in protein expression and particularly for the isolation of the polypeptide product. In particular, the invention relates to a method for preparing a polypeptide in such host cells.
Thus, in a first aspect, the invention provides a method for producing a polypeptide, comprising the steps of:
a) providing a bacterial host cell comprising an intracytoplasmic membrane, wherein the intracytoplasmic membrane has a composition which differs from other membranes in the host cell;
b) transforming the host cell with a nucleic acid construct comprising a coding sequence encoding the polypeptide and expressing the polypeptide in the bacterial host cell such that it becomes associated with the intracytoplasmic membrane; and c) isolating the polypeptide by separating the intracytoplasmic membrane from ?S other cellular components.
The composition of the intracytoplasmic membrane advantageously confers some degree of resistance to toxic effects of protein expression, particularly membrane protein expression.

The term "polypeptide" is used herein synonymously with "protein" and refers to any polypeptide of two amino acid residues or more in length. Protein expression means the production of a polypeptide from a nucleic acid encoding it, by a process of transcription (if necessary) and translation.
A host cell according to the invention may be any cell which is capable of harbouring a recombinant nucleic acid molecule, whether stably or transiently. Preferably, a host cell according to the present invention is a bacterial cell such as an E. coli cell.
Examples of suitable host cells are discussed in more detail below.
The composition of intracytoplasmic membranes according to the present invenrion differs from the membrane composition of a wild-type cell. The presence of a difference preferably refers to the differences in quantities and/or qualities of components of the lipid-containing fraction of the cell. This is explained in more detail below. The different membrane composition is referred to herein as an "altered"
membrane composition. The host cells disclosed herein comprise intracytoplasmic membranes which have an altered membrane composition.
The altered membrane composition of membranes according to the invention may be characterised by analytical techniques known to those skilled in the art and described herein below. Preferably, a membrane according to the invention may have one or more properties selected from; a lipid:protein ratio of greater than about 0.4; a membrane phospholipid composition comprising greater than about 4%
cardiolipin; a membrane phospholipid composition comprising less than about 20% phosphatidyl glycerol, or a membrane phospholipid composition comprising lower than average levels of phosphatidylethanolamine.
Toxic effects of protein expression in host cells may manifest themselves in numerous different ways, which may include impaired growth, loss of viability, morphological defects or other effects which are known to those skilled in the art, and are discussed in more detail below.

It is shown herein that the toxic effects of protein expression in host cells may be to some extent alleviated by the expression of at least part of subunit b or subunit c of ATP synthase. It is further disclosed herein that the expression of subunit b or subunit c of ATP synthase surprisingly contributes to the alteration of membrane composition in host cells according to the present invention. Thus, the invention provides that intracytoplasmic membrane production is induced by expression, in the host cell, of the b and/or c subunit of ATP synthase, or a fragment thereof capable of inducing membrane proliferation.
A nucleic acid construct for use in the methods of the invention may be prepared using recombinant DNA techniques known to those skilled in the art and discussed below.
Briefly, a construct for use in protein expression may contain a nucleotide sequence which encodes the protein to be expressed (termed a 'coding' sequence, or open 1 S reading frame (ORF)), and may also contain a nucleotide sequence which promotes, directs or drives the expression of said coding sequence (referred to herein as a "promoter"). Other elements may also be present in the nucleic acid construct;
examples of these are given below.
'Subunit b or subunit c of ATP svnthase' preferably refers to subunit b or subunit c from the Fo membrane sector of E. coli ATP synthase (Walker et al., 1982 Nature vo1.298, pp867-869). In a highly preferred embodiment, this refers to subunit b of the Fo membrane sector of E. coli ATP synthase, or a part thereof, examples of which are described herein.
Expression of a polypeptide of interest in host cells may be conveniently directed by a nucleic acid construct. Therefore, the present invention provides a host cell comprising a nucleic acid construct, said construct being capable of directing the expression of a polypeptide of interest, and at least a part of subunit b or subunit c of ATP synthase. In a highly preferred embodiment, expression of the polypeptide of interest and the expression of at least a part of subunit b or subunit c of ATP synthase are directed from a single nucleic acid construct.
The properties of the host cells) disclosed herein are particularly advantageous for the 5 expression/production of membrane proteins. Membrane proteins are any proteins which associate with or are capable of associating with the membrane fraction of cells.
As disclosed herein, the production of membranes having an altered membrane composition can be induced by expression of at least a part of subunit b or subunit c of ATPase in a host cell. This altered membrane composition may take the form of increased intracytoplasmic membrane production.
Advantageously the expression of at least part of subunit b or subunit c of ATP
synthase and the expression of the polypeptide of interest are directed from a single nucleic acid construct. Preferably, the expression of at least part of subunit b or subunit c of ATP synthase and the expression of the polypeptide of interest occurs simultaneously. In a preferred embodiment, the polypeptide of interest may be a heterologous or an endogenous membrane protein.
Simultaneously means that the expression of the polypeptide of interest and subunit b or subunit c of ATP synthase may temporally overlap. In some embodiments, expression of subunit b or subunit c of ATP synthase may precede expression of the polypeptide of interest. In this case, their expression will be considered to overlap if at a particular timepoint, both proteins are present in the same host cell.
Advantageously, in a vector according to the invention, the coding sequence encoding ATP synthase, or a fragment thereof, is placed promoter-proximal to (e.g.
upstream of) the coding sequence encoding the polypeptide and the two coding sequences are expressed in tandem from the same promoter.

Preferably, the promoters) employed in a vector according to the invention is an inducible promoter.
Induction of intracellular membrane production preferably occurs prior to, or simultaneously with, the production of the polypeptide in the host cell. This ensures that the intracellular membranes are present during polypeptide expression.
The present invention is useful for the expression of polypeptides, said expression being directed by any of numerous suitable methods known to those skilled in the art and described herein. For example, the expression system may make use of the bacteriophage T7 RNA polymerise system. Accordingly, the invention provides a method for expressing a protein in a host cell wherein the host cell express a bacteriophage RNA polymerise and wherein the expression system comprises a promoter sequence recognised by the polymerise. Preferably the polymerise is RNA polymerise.
Commonly available systems employing this method of expression include the pET
or pMW7 expression systems. Therefore, the invention provides a method for expressing a protein in a host cell wherein the expression system comprises the expression vector pET or pMW7.
It is often desirable to label an expressed protein. This labelling may be accomplished by any suitable means known to those skilled in the art, for example by expressing the polypeptide as a fusion protein with a detectable moiety such as Green Fluorescent Protein (GFP). Accordingly, 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 which serves as a detectable label. Preferably the detectable label is Green Fluorescent Protein.
Transforming may include transfecting, infecting, transducing, or otherwise introducing the nucleic acid into the host cell, and is also intended to cover transient trasfection or transformation with a non-replicating vector ('suicide vector') as well as stable transformation either with naked nucleic acid or with particles harbouring nucleic acid. The term vector as used herein includes plasmids, cosmids, gene cassettes as well as organismal vectors such as bacteriophage or the like.
Control sequences are nucleotide sequences which induce, enhance, promote, or in some way affect expression of the polypeptide of interest.
Polypeptides expressed according to the invention may be isolated by separating the intracytoplasmic membrane from other cellular membranes. Preferably, the separation is carried out by centrifugation. Membrane fractions may be prepared from the host cells comprising membranes having an altered membrane composition as described herein without the need for a high-speed centrifugation step as is required by prior art membrane preparation procedures. As disclosed herein, centrifugation at low speeds, such as 2,500-10,000 x g, is sufficient for the separation of membrane from cytoplasmic fractions of host cells according to the invention. An advantage of this procedure is that the intracytoplasmic membranes having the altered composition are also separated from other cellular membranes, which must be spun down at higher speeds.
The polypeptide being prepared may be a membrane protein. In another embodiment, the membrane protein may be a polypeptide fused to a membrane targeting protein.
A further embodiment of the invention provides a method for producing membrane proteins for use in screening assays. Screening of agents) for their ability to associate with or bind to membrane polypeptides may require the preparation and/or expression of said polypeptides. Therefore, the invention also relates to a method of screening agents which bind to, affect or modulate a desired membrane protein, comprising the steps of transforming the host cell as described herein with a vector according to the invention;
inducing expression of the desired membrane protein; culturing the host cells to produce the desired membrane protein; immobilising cell membranes on a support and exposing the membranes to the agent to be screened under conditions which promote the interaction of the agent with the polypeptide.
As used herein, the term agent refers to any entity which is lrnown or suspected of associating, either reversibly or irreversibly, with a polypeptide such as a membrane protein. Supports upon which cell membranes could be immobilised are well lrnown in the art, and include nitrocellulose based filters, supported filters or activated or coated surfaces such as ELISA plates.
It may be desirable to perform polypeptide screening on whole cells, without fractionating the host cells during or after polypeptide expression according to the invention. Thus, in a fiirther embodiment, the invention provides a method of screening agents which bind to, affect or modulate a desired polypeptide, comprising the steps of transforming a host cell as described herein with a vector according to the invention;
1 ~ inducing expression of E. coli F-ATPase subunit b or subunit c from the first expression unit, and culturing the host cells such that membrane production is induced;
inducing expression of the desired membrane protein from the second expression unit and culturing the host cells to produce the desired membrane protein; immobilising the cells on a support and exposing the cells to the agent to be screened under conditions which promote the interaction of the agent with the polypeptide.
Expression of polypeptides having a toxic effect may be improved by culturing the host cells at lower than usual temperatures. An example of a usual growth temperature for an E. coli cell is 37°C. Thus, the invention relates to a method of expressing a polypeptide in a host cell wherein said host cells) are cultured at 25°C.
Detailed Description of the Invention The term protein means any polypeptide of two or more amino acid residues or more in length. The terms protein and polypeptide may be used interchangeably herein, and may include, among other things, post-translationally modified polypeptides or polypeptides incorporating artificial amino or immino acid residues, amino acid analogues and the like.
Host Cells A host cell according to the invention may be any cell capable of harbouring, whether stably or transiently, a nucleic acid molecule capable of directing the expression of a polypeptide. Preferably, a host cell according to the present invention is a bacterial cell such as an E. coli cell. More preferably, a host cell according to the present invention is or is derived from 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). The term 'derived from' in the context of E. coli strains means that the a host cell may be genetically closely related to one or more of the strains set out herein. For example, an E. coli strain would be considered to be 1 ~ derived from another strain if the former could be arrived at from the latter by straightforward genetic manipulations known to those skilled in the art.
Toxicity The terms "toxic", "deleterious" and "toxic effect" are relative terms which can be understood without difficulty in the art. For example, Studier et al (1990) refer to the problem of genes whose product is toxic to host cells without the necessity to define this term in further detail.
In general however toxicity may be manifested by a variety of effects on the cell, including impaired cell growth, decreased copy number, an increase in cells in the growth media lacking the plasmid (Studier et al., 1990), filamentation of bacterial cells (George et al, 1994), induction of the SOS response (Murli & Walker, 1993) and/or ribosomal disruption (Dong et al, 1995).
Vectors A nucleic acid of the invention encoding a polypeptide of interest can be incorporated into vectors for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA
5 amplification or for DNA expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, 10 one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.
Both expression and cloning vectors generally contain nucleic acid sequence that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2~ plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, polyoma, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells.
Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another class of organisms for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be replicated by insertion into the host genome. However, the recovery of genomic DNA encoding a polypeptide of interest is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise DNA encoding a polypeptide of interest. DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.
Selectable Markers Advantageously, an expression and cloning vector may contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.
As to a selective gene marker appropriate for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene. Suitable markers for yeast are, for example, those conferring resistance to antibiotics 6418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2, TRP1, or HIS3 gene.
Since the replication of vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript~ vector or a pUC
plasmid, e.g. pUCl8 or pUCl9, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.
Promoters Expression and cloning vectors usually contain a promoter that is recognised by the host organism and is operably linked to nucleic acid encoding a polypeptide of interest.
Such a promoter may be inducible or constitutive. The promoters are operably linked to DNA encoding a polypeptide of interest by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Many heterologous promoters may be used to direct amplification and/or expression of a polypeptide of interest. The term "operably linked" refers to the components in a relationship permitting them to 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.
Promoters suitable for use with prokaryotic hosts include, for example, the [3-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding a polypeptide of interest, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding a polypeptide of interest.
Preferred expression vectors are bacterial expression vectors which comprise a promoter of a bacteriophage such as phagex or T7 which is capable of functioning in the bacteria. In one of the most widely used expression systems, the nucleic acid encoding the fusion protein may be transcribed from the vector by T7 RNA
polymerase (Studier et al, Methods in Enzymol. 185; 60-89, 1990). In the E.
coli BL21(DE3) host strain, used in conjunction with pET vectors, the T7 RNA
polymerase is produced from the 7~-lysogen DE3 in the host bacterium, and its expression is under the control of the IPTG inducible lac UV5 promoter. This system has been employed successfully for over-production of many proteins. Alternatively the polymerase gene may be introduced on a lambda phage by infection with an int- phage such as the CE6 phage which is commercially available (Novagen, Madison, USA). other vectors include vectors containing the lambda PL promoter such as PLEX (Invitrogen, NL), vectors containing the trc promoters such as pTrcHisXpressTm (Invitrogen) or pTrc99 (Pharmacia Biotech, SE) , or vectors containing the tac promoter such as pKK223-3 (Pharmacia Biotech) or PMAL (new England Biolabs, MA, USA).
Suitable promoting sequences for use with yeast hosts may be regulated or constitutive and are preferably derived from a highly expressed yeast gene, especially a Saccharomyces cerevisiae gene. Thus, the promoter of the TRP1 gene, the ADHI
or ADHII gene, the acid phosphatase (PH05) gene, a promoter of the yeast mating pheromone genes coding for the a- or a-factor or a promoter derived from a gene encoding a glycolytic enzyme such as the promoter of the enolase, glyceraldehyde-3-phosphate dehydrogenase (GAP), 3-phospho glycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase or glucokinase genes, the S. cerevisiae GAL 4 gene, the S.
pombe nmt 1 gene or a promoter from the TATA binding protein (TBP) gene can be used. Furthermore, it is possible to use hybrid promoters comprising upstream activation sequences (U AS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene, for example a hybrid promoter including the UAS(s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene (PH05-GAP
hybrid promoter). A suitable constitutive PH05 promoter is e.g. a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) such as the PH05 (-173) promoter element starting at nucleotide -173 and ending at nucleotide -9 of the PH05 gene.
An expression vector includes any vector capable of expressing nucleic acids encoding a polypeptide of interest that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector, that upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those with ordinary skill in the art and include those that are replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
Recombinant DNA Techniques Construction of vectors according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion.
Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function are known to those skilled in the art. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe which may be based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.
Recombinant polypeptides Recombinant polypeptides producible in cells according to the invention by the methods described herein include, but are not limited to, chymosin, insulin, an interferon, an insulin-like growth factor, an antibody including a humanised antibody, or a fragments thereof. Particularly preferred, however, are membrane proteins of prokaryotic and eukaryotic origin, including receptor proteins, chaperone proteins and fragments thereof, proteins of medical and pharmaceutical utility, nucleases and other enzymes useful as research tools, proteins involved in food processing, including brewing and vinification, in detoxification and in degradation of industrial and domestic waste.
It is observed that polypeptides expressed in host cells according to the invention are more likely to remain soluble than would otherwise be the case. In particular, polypeptides which are only partially soluble in unselected host strains, such as BL21 (DE3), are more soluble or completely soluble when produced in cells according to the invention.
The temperature of the culture conditions is an important factor in determining polypeptide solubility. Preferably, therefore, the culture is carried out at a reduced temperature. Advantageously, cells are cultured at between about 30 and about 20°C, most preferably at about 25°C.
A particularly preferred category of recombinant polypeptides which may be produced 10 by the method of the invention includes membrane proteins. Hitherto, such proteins have been difficult to produce in culture, especially bacterial cell culture, due to their toxicity. Moreover, in conventional expression systems, membrane proteins are not efficiently inserted into membranes on synthesis and are thus rarely functional. The expression system of the invention, however, provides an efficient system for membrane 15 protein expression.
Membrane Composition As mentioned above, altered membrane composition of a host cell means a membrane composition which differs in some way from the membrane composition of a wild type cell. The membrane composition refers to the quantities and/or qualities of the lipid-containing fraction of the cell. The composition would be considered to be 'altered' if it differs from that of a wild-type cell. The differences) may be in the chemical composition of the lipid fraction, or the differences) may be in the ratios of the various lipid-containing components to one another, or the differences) may be in the ratios of the lipid fraction to another cell fraction, for example to the protein fraction, or to the nucleic acid fraction, or to any other cellular fraction.
If the membrane composition of a host cell is in some way different to that of a wild-type cell, this would be considered to be 'altered'.
The altered membrane composition of host cells according to the invention may be characterised by analytical techniques known to those skilled in the art and described herein below. Preferably, a host cell according to the invention may have an altered membrane composition as judged by one or more properties selected from;
altered lipid:protein ratio, altered membrane phospholipid composition with respect to one or more of cardiolipin, phosphatidyl glycerol or phosphatidylethanolamine; or any other membrane or lipid related property known to those skilled in the art.
A host cell would be considered to have altered membrane phospholipid composition if it had a lipid:protein ratio of greater than about 0.4, preferably greater than 0.414, more preferably greater than about 0.5, more preferably greater than about 0.6, yet more preferably greater than about 0.7 or even more.
A host cell would be considered to have altered membrane phospholipid composition if it had a membrane phospholipid composition comprising greater than about 2-4%
cardiolipin, preferably greater than 4.3%, preferably greater than 5%, preferably greater than 6%, preferably greater than 8%, preferably greater than 10%, more preferably greater than 11%, yet more preferably greater than 11.3%
cardiolipin, or even more.
A host cell would be considered to have altered membrane phospholipid composition if it had a membrane phospholipid composition comprising less than about 20%
phosphatidyl glycerol, preferably less than 19 %, preferably less than 17%, preferably less than 15%, more preferably about 13.2% phosphatidyl glycerol, or even less.
A host cell would be considered to have altered membrane phospholipid composition if it had a membrane phospholipid composition comprising lower than average levels of phosphatidylethanolamine. Average levels of phosphatidylethanolamine may be about 76.6% phosphatidylethanolamine. Preferably a. host cell according to the invention may have a membrane phospholipid composition of less than 76.6%
phosphatidylethanolamine, preferably 75.45% phosphatidylethanolamine or even less.

Further, altered membrane composition may also mean that lipid composition ratios remain substantially the same, but that membrane morphology is altered. In this case, membrane composition would be considered to be altered if it was morphologically different from a wild-type cell. Morphological differences which are shown herein for host cells having altered membrane composition include presence of one or more characteristics such as vesicles, inclusion bodies, tube-like structures or networks, or lamellar proliferation. Other altered membranal morphologies are known to those skilled in the art.
Subunits of ATP Synthase As disclosed herein, subunit b or subunit c from the Fo membrane sector of E.
coli ATP synthase (Walker et al., 1982 Nature vo1.298, pp867-869 (subunit b);
Fillingame (1996) Curr. Op. Struct. Biol. vol.6 pp491-498 (subunit c)) are useful in the preparation of host cells according to the invention. In a highly preferred embodiment, subunit b of the Fo membrane sector of E. coli ATP synthase, is used in preparing host cells according to the invention. In a further embodiment, the invention relates to the use of subunit b or a part thereof, in the preparation of host cells with altered membrane composition according to the invention.
Parts) of subunit b which are useful in the present invention include but are not limited to the entire subunit b, amino acids 1-157 of subunit b, amino acids 1-25 of subunit b, amino acids 1-34 of subunit b, amino acids 1-48 of subunit b, amino acids 25-157 of subunit b, or any other part or combination of parts of subunit b which produces altered membrane composition in a host cell as described herein.
Similarly, parts of subunit c which are useful in the present invention include but are not limited to the entire subunit c, or fragments) thereof.
Membrane Preparation It is an advantageous feature of the present invention that membrane fractions may be easily prepared from host cells as disclosed herein. Preparation of membranes from prior art cells for polypeptide expression requires a high speed centrifugation step at 100,000xg or even more. This step is not only time consuming, often requiring many hours at the high speed stage, but is also laborious. Samples must be prepared and syringed into single-use tubes which must then be meticulously balanced to within a fraction of a gram before being sealed ready for centrifugation. The centrifugation itself requires a sophisticated ultracentrifuge with evacuation, which is often a major expense, both in terms of initial cost and of ongoing operation and maintenance. In contrast, membranes may be prepared from host cells according to the present invention without the need for such high-speed centrifugation. The altered membranes of the host cells of the present invention may be conveniently prepared utilising only low speed centrifugation of about 2,500-10,000xg. This is much less costly, considerably quicker and substantially more convenient than preparing membranes according to prior art methods. This is discussed in more detail in the Examples section.
Screening The polypeptide may be any polypeptide for which it is desired to identify an interaction with the agents to be screened. However, membrane proteins, in particular receptor proteins, are particularly indicated.
Membranes may be obtained from disrupted cells, from which they can be easily isolated, for example by centrifugation, or may be in the form of intact cells. Part of the membrane fraction from a disrupted cell according to the invention is obtained in the form of liposome-like vescicles, which may be immobilised on liposome-specific supports such as that available from Biacore.
If necessary, the phospholipid levels in the membranes may be adjusted to mimic the levels present in the natural environment of the polypeptide to be screened.
Methods for surveying ligand binding to membrane proteins are well known in the art.
The main techniques used for separation of the free ligand from the bound ligand include rapid filtration, centrifugation, dialysis, gel filtration, precipitation or absorption. Alternatively, the "liposomes" containing the polypeptide to be screened are bound to a support compatible with the Biacore system. A library of ligands is generated and ligands are screened for their ability to bind the polypeptide of interest.
Ligands having a high binding constant to the polypeptide of interest are analysed further in vitro.
Brief Description of the Figures Figure 1 shows a graph (panel A), and Coomassie stained polypeptides (panels B
and C) indicating the effect of the expression of E. coli F-ATPase subunit b on the growth of E. coli C43(DE3). Fresh colonies of host cells containing the plasmid for over-expression of subunit b are inoculated into 2 x TY medium supplemented with ampicillin. The cultures are grown at 37°C for 4-5 h, and when the O.D.
at 600 nm of the cultures reaches 0.6, IPTG (0.7 mM) is added. Then the cells are grown either at 37°C or 25°C. Panel A, growth of C43 (DE3) cells at (o) 25°C and (~) 37°C. Panels B
and C, analysis by SDS-PAGE of 10 p.1 samples of C43 (DE3) cultures grown at 37°C
and at 25°C, respectively. Subunit b is the band at about 20 kDa.
Figure 2 shows Coomassie stained polypeptides indicating the co-expression of subunits b and c of E. coli F-ATPase in E. coli C43(DE3). Their expression from plasmids pEc.bc6 (bc) and pEe.cbS (cb) is compared. Freshly transformed colonies C43(DE3) are inoculated into 2 x TY medium (50 ml) supplemented with ampicillin.
The cultures are grown at 37°C for 4-Sh, and when their O.D. at 600 nm had reached 0.6, IPTG (0.7 mM) is added. Then the cells are grown at 37°C for 1, 2 or 3 h. The equivalent of 20 ~l of culture is analysed by SDS-PAGE at the times indicated above each slot. In the centre lane, 1 ~g of E. coli F,F°-ATPase (ATP
synthase) is loaded as a marker. The positions of its various subunits are shown on the right.
Figure 3 shows electron micrographs of thin sections of E. coli cells over-producing subunit b of E. coli ATP synthase. Panels A and B, C41 (DE3) cells over-producing subunit b grown at 37°C or 25°C after induction of expression for 3h or 18h, respectively. Panels C and D, C43(DE3) cells over-producing subunit b, 3 h and 18 h after induction at 37°C or 25°C, respectively. Panels E and F, C41(DE3) and C43(DE3) cells containing plasmid without insert, 4 h after addition of IPTG
to 5 cultures grown at 37°C. Panels G and H, C41(DE3) and C43(DE3) cells without plasmid grown under the same conditions. The scale bar represents 0.2 or 0.28 ~m in panels A-D and E - H, respectively.
Figure 4 shows Coomassie stained polypeptides indicating protein contents of 10 proliferated membranes isolated from E. coli C43(DE3) cells over-producing subunit b. After induction of expression, the cells are kept at 25°C for 18 h and samples are analysed by SDS-PAGE. Lane (a), total cell extract (10 ~l); lane (b), low speed pellet (3,000 x g; 30 ~g of protein); lane (c), membrane fraction from high speed centrifugation (100,000 x g ) of the supernatant from lane (b) (35 ~.g of protein); lane 15 (d), high speed pellet obtained by resuspension and washing of material in lane (b) (20 ~g of protein). Lanes (1-11), fractions from sucrose step gradient fractionation of material in lane (d) (5 ~l samples from 1 ml fractions).
Figure 5 shows Coomassie stained polypeptides indicating protein contents of altered 20 membranes isolated from E. coli C43 (DE3) cells over-expressing subunits b and c of E. coli ATP synthase. Samples are analysed by SDS-PAGE. Lanes (a) and (b), low speed pellets (3,000 x g) (25 pg of protein) from cells over-producing subunit b only and subunits b and c together (genes in the be configuration), respectively.
Lane (c), high speed pellet (100,000 x g) (35 ~g of protein) from cells over-expressing subunit c only. The intense band with an apparent molecular weight of 16 kDa is identified as the heat-shock protein Hsp 16 by protein sequencing.
Figure 6 shows electron micrographs in negative stain of isolated proliferated membranes. The membranes are obtained at low speed centrifugation (3,000 x g) from E. coli C43(DE3) host cells over-expressing subunit b disrupted either in a French press (panel A), or by osmotic shock after treatment with EDTA and lysozyme (panel B). The former membranes are fractionated by sucrose gradient, and analysed by negative stain electron microscopy. Panel C, fraction 10 from the sucrose gradient (see Fig. 3, buoyant density 1.18 g/ml). Panel D, fraction 7 from the sucrose gradient (see Fig. 3, buoyant density 1.10 g/ml). (scale bar 0.28 p.m).
Figure 7 shows Coomassie stained polypeptides resulting from trypsinolysis of subunit b of E. coli ATP synthase incorporated into altered membranes. The supernatants (panel A) and pellets (panel B), respectively, obtained by trypsin digestion for the periods indicated, are analysed by SDS-PAGE. Bands with MT
of 12, 8, 7 and 6 kDa observed in the supernatant after 5 min of digestion (indicated by the arrows on the left in panel A) have the same N-terminal sequence, from residue onwards. In panel B, the band indicated by an arrow with Mr of 7 kDa corresponds to residues 1-36 of the subunit b.

EXAMPLES
EXAMPLE 1: OVER-EXPRESSION OF E. COLI ATP SYNTHASE B AND C
SUBUNITS IN HOST CELLS
In order to produce host cells with altered membrane composition suitable for use in polypeptide expression, subunit b and c of ATP synthase are expressed in E.
coli host strains C41(DE3) and C43(DE3).
The host strains E. coli C41(DE3) and C43(DE3) have been described previously (Miroux and Walker, 1996).
The unc E and unc F genes, encoding E. coli ATP synthase subunits c and b, are amplified by PCR from E. coli DNA. Each gene is cloned separately into the expression plasmid pMWI72 (Way et al., 1990), giving rise to expression plasmids pMW 172(Ecc) and pMW 172(Ecb), respectively.
The insert of E. coli cb is generated by PCR using the forward primer TAG GAA TTC ATA TGG AAA ACC TGA ATA TGG ATC TGC TGT
which contains sites for the restriction enzymes Eco RI and Nde I, the reverse primer CGA AAG CTT TTA TTA CAG TTC AGC GAC AAG TTT ATC CAC G
which contains a Hind III site and E. coli cells as template. The PCR product is cloned into the Eco RI and Hind III sites of M 13 and is checked by sequencing. Rf M

DNA is grown and purified from this clone and the insert is excised with Nde I
and Hind III and ligated into the expression vector PMW172.

The construct for E. coli be has the intracistronic sequence AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA
S between the DNA sequence for the b and c subunits. This is also made by PCR
of two individual reactions, one for each subunit, and a third reaction to join the products from the first two. Reaction l, for the b subunit, is carried out with the forward primer TAG GAA TTC ATA TGA ATC TTA ACG CAA CAA TCC TCG GCC
which incorporates restrictions sites for Eco RI and Nde I and the reverse primer ATC TCC TTC TTA AAG TTA AAC AAA ATT ATT TTA CAG TTC AGC GAC
AAG TTT ATC
which contains sequence for a ribosome binding site. Reaction 2, for the c subunit, is performed using the forward primer TTT GTT TAA CTT TAA GAA GGA GAT ATA ATG GCT TCA GAA AAT ATG
ACG CCG
which contains the same sequence for a ribosome binding site and the reverse primer CGA AAG CTT TTA CTA CGC GAC AGC GAA CAT CAC GTA CAG
which incorporates the restriction site for Hind III. Both of these reactions use E. coli cells as template. The third PCR is carried out using the purified product and forward primer from reaction 1 and the purified product and reverse primer from reaction 2.
The final product is digested with Nde I and Hind III and cloned directly into pMW
172.

Segments of unc F encoding amino acids 1-25, 1-34, 1-48 and 26-157 of subunit b (see following Examples) are cloned into pMWI72 yielding plasmids pEc.bN25, pEc.bN34, pEc.bN48 and pEc.b132C, respectively. Vectors for co-expression of subunits b and c in pMWI72 are made with subunit b promoter proximal (pEc.bc6), and with subunit c promoter proximal (pEc.cb4 and pEc.cbS, respectively. Plasmid pEc.cb4 contains the natural intercistronic sequence (Walker et al., 1984), whereas in pEc.cbS a ribosome binding site is inserted between the genes.
Protein over-expression and harvest of host cells Bacteria are grown at 37°C in 2xTY medium (16 g/1 tryptone, 10 g/1 yeast extract, 5 g/1 NaCI, pH 7.4) to an optical density of 0.6 at 600 run. Then IPTG is added to a final concentration of 0.7 mM. The cells are grown for a further period at either 37°C or 25°C and then centrifuged (2,000 x g, 10 min). They are resuspended in deionized water, re-centrifuged and then resuspended in 20 volumes of TEP buffer (10 mM
Tris, pH 8.0, 1 mM EDTA and 0.001 % (w/v) phenylmethylsulphonyl fluoride). Growth on agar plates is achieved by preparing plates from liquid medium as above, with the addition of 1% agar as known to those skilled in the art.
Cultures of E. coli C43(DE3) grown at 37°C enter stationary phase 3-4 h after induction of expression of subunit b, and then the cell density decreases (Fig 1A), whereas the stationary phase cell density of cultures grown at 25°C
after induction of expression remains constant.
The viability of cells from both cultures is examined by plating them on TY
plates, in the absence and presence of ampicillin and IPTG, both singly and together (Miroux and Walker, 1996). Cells from the 37°C culture form large colonies on all types of plate, but the colonies from plates containing ampicillin lose the ability to express subunit b, demonstrating that the expression of subunit b at 37°C is deleterious.

In contrast, the sample from the 25°C culture forms small colonies in the presence of IPTG, and in liquid medium 90% of the population of these colonies retain the ability to express subunit b.
5 The kinetics of ATP synthase subunit expression are examined by monitoring the presence or absence of the appropriate protein(s). Protein concentrations are estimated by the bicinchoninic acid assay (Pierce Chemicals, Rockford, IL). Proteins are analysed by SDS-PAGE in 12-22 % gradient gels prepared and run in the buffers of Laemmli (1970). Amino-terminal sequences are determined with the aid of an Applied 10 Biosystems Procise model 494 protein sequencer. Peptides and proteins are examined by electro-spray ionization mass spectometry in a Perkin Elmer-Sciex API III+
triple quadrupole instrument.
At 25°C, the onset of production of subunit b is delayed by 2 h relative to 37°C, but 15 about the same final level of expression of subunit b is obtained at either growth temperature (compare panels B and C in Fig.l ).
Subunit c is over-expressed at high levels in both C41(DE3) and C43(DE3). At 37°C, the over-expression in C41(DE3) appears toxic, but less severe than the toxicity 20 associated with the expression of subunit b. The overgrowth of the C41(DE3) culture is overcome partially by growing the cells at 25°C. The culture reaches a final ODboo of 4.7, and contains twice as much (50 mg/1 of culture) of subunit c as the culture grown at 37°C. Similar to the pattern observed for subunit b, over-expression of subunit c in C43(DE3) at 25°C is delayed with respect to overexpression in C41(DE3).
25 In this particular case, the culture reaches a final OD6oo of about 6.3, and contains a slightly lower amount of the recombinant protein (about 30 mg / 1 of culture).
The co-expression of subunits b and c is examined with their genes in dicistronic arrangement. With the gene for subunit b promoter proximal (plasmid pEc.bc6), very high levels of over-production of both proteins in both mutant hosts are obtained. The growth is similar to cultures over-expressing subunit b only. With the gene for subunit c promoter proximal, as in the unc operon, and with the natural intercistronic sequence (Walker et al., 1984) (plasmid pEc.cb4), the expression of both proteins is low.
Insertion of a synthetic intercistronic sequence (plasmid pEc.cbS) improves the expression of both proteins (Fig. 2).
Thus, it is demonstrated how ATP synthase subunits b and c can be efficiently expressed in host cells. It is also shown how their expression can be optimised and enhanced. Further, it is taught how toxic effects of such overexpression can be overcome.
EXAMPLE 2: INDUCTION OF MEMBRANE PROLIFERATION IN HOST
CELLS
It is taught herein that altered membrane composition in host cells according to the invention can be obtained by the expression of subunits of ATP synthase.
Expression of said subunits is shown in Example 1 above. As explained above, altered membrane composition refers to one or more membranal characteristics, which characteristics include morphological characteristics.
ATP synthase subunits are expressed in host cells as in Example 1, and the effects on membrane morphology are examined.
Membranal structures are examined using electron microscopy. Bacteria are fixed first with 2% glutaraldehyde and washed twice with cacodylate buffer (50 mM, pH
7.2), and then with 4% osmium tetroxide, followed by three washing steps with Kellenberger buffer (Kellenberger et al., 1958). Pellets are embedded in 2%
agar, cut into 1 mm cubes, and stained in the dark with a 0.5% (w/v) uranyl acetate for 2 h. The samples are dehydrated with alcohol (60% to 100%), transferred to propylene oxide and propylene oxide/Epon mixtures and finally embedded in Epon 812. The resin is polymerised at 60°C for 2 days. Thin sections are cut, adsorbed on electron microscope grids coated with plastic films and stained with 2% uranyl acetate and lead citrate (Reynolds, 1963).
In other experiments, bacteria are disrupted either in a French press or by EDTA-lysozyme treatment and osmotic shock (Osborn et al., 1972). Supernatants and pellets are collected by low speed centrifugation (3000 x g) and then adsorbed on to copper grids coated with carbon, and stained negatively with 2% (w/v) uranyl acetate.
Samples of membranes from sucrose density gradients are treated similarly.
All samples are examined in a Philips CM12 transmission microscope operated at kV, and micrographs are recorded on Kodak film.
The over-expression of subunit b in C41(DE3) and C43(DE3) is accompanied by proliferation of internal structures (Fig. 3, panels A-D) that are not observed in controls, either harvested immediately before the induction of the expression (Fig. 3, panels E and F), or lacking the expression plasmids (Fig. 3, panels G and H).
The induced internal membranes in C41(DE3) are multi-lamellar structures, whereas in C43(DE3) at both 25°C and 37°C, cytoplasmic networks form. Under the conditions of maximal expression of subunit b, internal structure formation is more extensme m C43(DE3) than in C41 (DE3) (compare panels A and C in Fig. 3).
The toxic effect of the over-expression of subunit b in C41(DE3) and in C43(DE3) at 37°C for 18 h after induction is also evident in electron micrographs of the bacteria.
Many cells lyse, and lysis is accompanied by deposition of large amounts of extracellular debris. In contrast, almost all of the C43(DE3) cells over-producing subunit b at 25°C remain intact (Fig. 3, panel D), even after the formation of the internal networks. Proliferation of internal mufti-lamellar structures similar to those in Fig. 3 (panels A and B) accompanies over-production of subunit c in both C41(DE3) and C43(DE3).

Thus, it is demonstrated that preparing host cells according to the invention by expressing subunits of ATP synthase in E. coli strains such as C41(DE3) and C43(DE3) produces an altered membrane composition as explained above.
EXAMPLE 3: PREPARATION OF ALTERED MEMBRANES
The proliferated membranes from C43(DE3) over-expressing subunit b as in Example 2 are harvested by low speed centrifugation of broken cells from a culture grown at 25°C for 18 h after induction, and collection of the resulting pellet.
Membranes are prepared by harvesting bacteria, and disrupting the cells by passing the suspension twice through a French pressure cell at 4°C. In some experiments, harvested bacteria are disrupted by treatment with EDTA and lysozyme, followed by osmotic shock (Osborn et al., 1972; Yamato et al., 1978).
Membranes from E. coli C43(DE3) cells expressing subunit b that have been grown at 25°C for 18h after induction are collected by centrifugation at 2,500 x g. The pellet is freed from unbroken cells and cell debris by resuspension in the same volume of TEP
buffer and centrifugation at 10,000 x g. Otherwise, the 2,500 x g supernatant is centrifuged directly at 10,000 x g. The remaining supernatants are ultra-centrifuged for 3 h at 100,000 x g.
Washed membranes containing subunit b are suspended in TEP buffer at a concentration of 2.5 mg of protein/ml. Portions (1 ml) of this suspension are applied to the top of discontinuous sucrose gradients prepared in centrifuge tubes (14 x 95 mm) with a cushion (2 ml) of 50% (w/v) sucrose, overlayered with 30% (w/v) sucrose (3 ml), 10 % (w/v) sucrose (3 ml) and 5% (w/v) sucrose (2 ml), all dissolved in a buffer consisting of 20 mM Tris, pH 8.0, and 0.001 % phenylmethylsulphonyl fluoride.
Samples are centrifuged for 18 h at 155,000 x g.

Most of the over-expressed subunit b is associated with the low-speed pellet, and it comprises about 40% of the protein in the fraction. This level is raised to about 75-80% by a washing step (see Fig. 4, lanes b and d). The washed membrane fraction with the highest subunit b content (Fig. 4, lane 10) has a buoyant density of 1.18 g/ml on a sucrose density gradient. Small amounts of other fractions with lower contents of subunit b are also observed on these gradients, and an additional membrane fraction devoid of subunit b is obtained by ultracentrifugation.
A low speed membrane fraction is also obtained from C43(DE3) cells containing the co-expression plasmid pEc.bc6. In cells overexpressing subunit c only, no low speed pellet is obtained, and much of the overproduced subunit c appears to be in the high-speed fraction (100,000 x g) (Fig. 5).
Washed membranes containing subunit b have the appearance of tubes or ribbons linking large vesicles (Fig. 6, panel A), and similar structures are observed also in cells broken by EDTA-lysozyme treatment and osmotic shock (Osborn et al., 1972) (Fig. 6, panel B). The tubes (or ribbons) and vesicles have buoyant densities on a sucrose gradient of 1.10 g/ml and 1.18 g/ml, respectively. The vesicles contain more subunit b than the ribbons (Fig. 6, panels C and D). After EDTA-lysozyme treatment, the cells do not swell indicating that the intracytoplasmic network is not an extension of the inner membrane of E. coli.
Thus, the present invention provides a convenient method for preparation of the altered membranes of host cells as described herein. This method is particularly advantageous since it requires only low-speed (2,500-10,000 x g) centrifugation, thereby alleviating the need for expensive and time-consuming high-speed centrifugation. Further, the morphological characteristics of altered membranes of host cells according to the invention are demonstrated.

EXAMPLE 4: CHARACTERISATION OF HOST CELL MEMBRANES
Composition of internal membranes Under most growth conditions, bacteria maintain a constant lipid:protein ratio (Cronan 5 and Rock, 1984), and values of 0.4 are typical of E. coli inner membranes (Ingraham et al., 1983).
In cells of C41(DE3) and C43(DE3) which are not over-expressing subunit b, the ratio has a normal value (see Table 1), but in C43(DE3) cells over-expressing subunit b the 10 lipid content and the ratio value for the proliferated membranes are much higher. This higher lipid content is reflected in the high viscosity of the membranes. In contrast, the value reported for membranes of E. coli cells over-expressing fumarate reductase, which can be accompanied by internal membrane proliferation (Weiner et al., 1984), is close to the normal value.
1 S Table 1. Phospholipid contents of proliferated membranes accompanying over-expression of ATP synthase subunit b in E. coli C43(DE3).
Membrane Lipid: Phospholipid composition sample protein CL PG PE

low speed0.701 14.50.7 12.510.2 72.90.8 pellet high speed0.284 8.211.2 13.91.2 78.010.1 pellet C43(DE3) 0.376 2.00.7 20.31.5 77.71.6 control C41(DE3) 0.414 4.311.2 20.20.4 75.51.2 control The values are obtained from 3-4 independent determinations.

In order to further characterise the altered membranes of host cells according to the invention, membrane phospholipid analysis is carried out.
Lipids are extracted according to Bligh and Dyer (1959). To a portion of the membrane fractions, 1M HC1 (20 ~1) is added with water to a total volume of 2001.
Chloroform : methanol (1: 2.2, v:v; 640 ~.1) is added and the sample is vortexed for 1 minute. Then 0.1 M HC 1 (200 a 1 ) and chloroform (200 ~l) are added and the sample is again vortexed for 1 minute. The mixture is centrifuged, and the chloroform phase is removed. Chloroform (200 ~1) is added and mixed with the aqueous phase for 1 minute and then the mixture is centrifuged. The chloroform fractions are pooled and mixed with buffer (50 mM Tris-HCl, pH 8.25, 0.1 M NaCI, 0. 1 M EGTA). The mixture is centrifuged and the chloroform phase is removed and evaporated. The lipid extracts are hydrolysed in perchloric acid (300 ~1) at 180°C for 3 h and then the phosphorus concentration is determined (Rouser et al., 1970).
The phospholipid compositions of the lipid extracts of membrane fractions (150 nmol phosphorus) are analysed by thin layer chromatography (TLC) on silica 60 plates using chloroform : methanol: acetic acid, 65:25:10 (by vol.) as solvent. The lipids are visualised with iodine vapour, scraped off, boiled and centrifuged to remove traces of silica and the phosphorus content is determined as described above.
Phosphatidic acid (PA) and cardiolipin (CL) are not resolved in this system, but they are in two other systems; the first system is a 2-dimensional system with chloroform methanol : water : ammonia (68:28:2:2, by vol.) as solvent in the first dimension, and chloroform : methanol : acetic acid (65:25: 10, by vol.) in the second dimension.
In the second system, (Fine and Sprecher, 1982), phospholipids are separated on a silica plate pre-treated with 1.2 % boric acid, with chloroform : methanol :
water ammonia (120:75:6:2, by vol.) as solvent. No phosphatidic acid is found samples using either chromatographic system.

Phospholipids on silica plates are visualised by spraying with phosphorus reagent (molybdenum (N) oxide) and then heating until a blue colour appeared.
The phospholipid composition of an average E. coli cell is 76% phosphatidyl ethanolamine, 20% phosphatidyl glycerol with small amounts of cardiolipin and unidentified species (Ames, 1968; Raetz, 1978; Randle et al., 1969, Cronan, 1968).
The head group composition is unaffected by changes in growth conditions, but it does change during the growth cycle, phosphatidyl glycerol being converted partially to cardiolipin by condensation of two molecules of phosphatidylglycerol early in stationary phase (Cronan and Vagelos, 1972). It has been suggested that cardiolipin might help bacteria to survive when they are not actively growing and dividing, possibly by ensuring the minimal phospholipid content in the membranes and by stabilising the membrane structures (Hiraoka et al., 1993).
The membranes isolated from C41(DE3) and C43(DE3) cells in which subunit b was not being expressed contain about 2-4% cardiolipin, but in C43(DE3) over-expressing the subunit, the level rises dramatically in the proliferated membranes to about 14% at the expense its biosynthetic precursor, phosphatidyl glycerol which drops from about 20% in the controls to about 13% (see Table 1). Phosphatidylethanolamine levels in proliferated membranes are also slightly lower than in controls.
Cardiolipin has been proposed to have roles in transport of proteins through the membranes, and anionic phospholipids have been shown to modulate the insertion into membranes (Liu et al., 1997; van Klompenburg et al., 1997) and to act as chaperones 2~ in assembly of membrane proteins (Bogdanov et al., 1996). Thus, the observations reported here demonstrate the utility of host strains according to the invention in facilitating the process of membrane protein over-expression, folding and insertion into membranes.
It is unlikely that the increase in cardiolipin content accompanying subunit b over-expression is a consequence of a mutation in its biosynthetic pathway, because the levels in control cells are normal. It is more likely that the delay in the onset of protein over-production in C41(DE3) and C43(DE3) relative to BL21(DE3) improves not only the coupling between transcription and translation, but also between subunit b production and cardiolipin biosynthesis.
S
Subunit b is incorporated into internal membranes Subunit b of E. coli ATP synthase has a membrane spanning N-terminal hydrophobic segment, followed by a highly charged hydrophilic domain that protrudes from the membrane and interacts with the F~ catalytic domain (Walker et al., 1982). In isolated F°, the extrinsic domain of subunit b can be proteolysed, thereby preventing reassociation of FI (Perlin et al., 1983).
Therefore, in order to investigate whether over-expressed subunit b had been incorporated correctly into the proliferated membranes of host cells according to the invention, its topography in the isolated membranes is explored by trypsinolysis.
Membranes isolated from E. coli C43(DE3) cells grown at 25°C in which subunit b had been overproduced (see above) are suspended in TEP buffer at a concentration of 2.5 mg of protein/ml. Trypsin (1:20, w/v) is added and the suspension is kept at 30°C
for 1 h. Samples are removed at intervals, and proteolysis is terminated by addition of soybean trypsin inhibitor (5-fold excess by wt). The course of proteolysis is monitored by SDS-PAGE analysis of the fragment polypeptides. The samples are cooled to 4°C, and the membranes are harvested by centrifugation at 128,000 x g for 1 h, dissolved in 6M guanidine hydrochloride and peptides are isolated by reverse-phase chromatography on a C8 Aquapore RP-300 column (7 ~m particles, 300 A pore size, 100 mm x 2.1 mm i.d.; from Applied Biosystems, Warrington, Cheshire, U. K.).
The column is equilibrated in 0. 1 % trifluoroacetic acid and eluted with a linear gradient acetonitrile. The peptides are analysed by electrospray ionization mass spectometry, and, after separation by SDS-PAGE, transfer to a poly(vinylidenedifluoride) membrane, and staining with PAGE Blue 83 dye, by N-terminal sequence analysis.

After digestion for S minutes, four major proteolytic fragments with apparent Mr values of 12, 8, 7 and 6 kDa are detected in the soluble fraction (Fig. 7A).
They all have the N-terminal sequence SQILDEKAE.... corresponding to residues 83 onwards.
Analysis by SDS-PAGE of the peptides in the pellet fraction (Fig. 7 B) reveals a fragment with an apparent Mr of 7 kDa. This fragment stains poorly with Coomassie blue dye, indicating that it could be hydrophobic. Its N-terminal sequence is that of intact subunit b, and its molecular mass measured by electrospray-ionization mass spectrometry is 4113.3, corresponding to residues 1-36 of subunit b (calculated molecular mass 4115.1), and arising by tryptic cleavage after arginine-36.
There is no evidence for cleavage after lysine-23, which is consistent with the region around this residue being protected from trypsinolysis by being in a membrane associated a-helix as suggested by the NMR structure (Dimitriev et al., 1999).
These characteristics are consistent with subunit b having been inserted correctly into the membrane, thus demonstrating that host cells according to the invention provide for correct folding and processing of polypeptides expressed therein.
EXAMPLE 5: PRODUCTION OF DIFFERENT MEMBRANE
CHARACTERISTICS BY EXPRESSION OF DIFFERENT FRAGMENTS OF
ATPASE SUBUNIT(S) In order to investigate the requirements for the proliferation of internal membranes in E. coli, truncated forms of subunit b are introduced into C41(DE3) and C43(DE3) on expression vectors under the same conditions used for the intact protein.
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 membrane spanning part of subunit b. When peptide 1-25 or 1-34 is expressed in C41(DE3), inclusion bodies are observed in electron micrographs of cioss-sections of cells, and no internal membranes are observed when they are expressed in C43(DE3).

Expression of peptide 1-48 induces the formation of vesicular structures in C41(DE3) and inclusion bodies in C43(DE3). Expression of residues 25-157, lacking the hydrophobic domain of subunit b, is accompanied by less membrane proliferation than with the intact protein in both C41(DE3) and C43(DE3), with different morphology 5 (see Table 2).
Table 2. Effect of over-expression of subunit b of E. coli F-ATPase and truncated forms on internal membrane proliferation in E. coli C41(DE3) and C43(DE3).
Subunit b and Appearance in fragments electron microscope C41(DE3) C43(DE3) 1-157 vesicles network of tubes 1-25a inclusion bodiesno proliferation 1-34a inclusion bodiesno proliferation 1-48 few vesicles inclusion bodies 25-157 vesicles vesicles 10 athe expression of these fragments in C43(DE3) appears very low /
undetectable.
This demonstrates the utility of expressing subunit b peptides or fragments thereof according to the invention in order to produce host cells with altered membrane composition such as altered membrane morphology.
EXAMPLE 6: EXPRESSION OF POLYPEPTIDES IN HOST CELLS OF THE
INVENTION
Heterologous over-production of membrane proteins associated with internal membrane proliferation has been observed with bacterial light-harvesting complexes in Rhodobacter spheroides (Fowler et al., 1995), over-expression of PMA2 ATPase in Saccharonrvces cerevisiae. (Supply et al., 1993) and others (Ohkuma et al., 1995;
Gong et al.. 1996) are accompanied by intracellular proliferation of membranes. Some of these systems have been suggested as general tools for over-expressing recombinant integral membrane proteins (Fowler et al., 1995; Gong et al., 1996). However, the wide-spread and well known genetics of E. coli makes this organism an ideal host for membrane protein over-production.
Bacterial over-expression of membrane proteins can be improved by optimisation of the host system. In order to over-produce a membrane at high levels transcription and translation should remain coupled as should the synthesis of phospholipids and the synthesis, folding and insertion of the membrane proteins.
In the case of over-production of subunit b in C43(DE3), incorporation of the protein in a membrane soluble form leads to formation of a network of tubular structures.
Both phospholipid composition and the structure of the recombinant protein appear to influence the formation of these structures.
Similar effects accompany co-expression of subunits b and c providing that the gene for subunit b is promoter proximal, but not vice versa.
Thus, it is possible to over-express membrane proteins and to arrange for their folding and membrane insertion by expressing them in tandem with subunit b in host cells according to the invention.

REFERENCES
Ames, G.F. (1968). Lipids of Salmonella typhimurium and Escherichia coli:
structure and metabolism J. Bacteriol. 95, 833-843.
Bogdanov, M., Sun, J., Kaback H. R. & Dowhan W. (1996). A phospholipid acts as chaperone in assembly of a membrane transport protein. J. Biol. Chem. 20, 1161511618.
Bligh E. G. & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911.
Cronan J. E.(1968). Phospholipid alterations during growth of Escherichia coli. J.
Bacteriol. 95, 2054-2061.
Cronan J. E. & Vagelos P. R. (1972). Metabolism 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. In Escherichia coli and Salmonella typhimuriun Neidhart 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). Structure of the membrane domain of subunit b of the Escherichia coli FoF~ ATP synthase. J. Biol. Chem. 274, 15604.
Dong, H., Nilsson, L. & Kurland, C. G. (1995). Gratuitous overexpression of genes in Esclzerichia coli leads to growth inhibition and ribosome destruction. J.
Bacteriol.
177, 1497-1504.

Fillingame, R. H. (1996). Membrane sectors of F- and V-type H+-transporting ATPases. Curr. Op. Struct. Biol 6: 491-498.
Fine, J. B. & Sprecher, H. (1982). Unidimensional thin-layer chromatography of pliospholipids 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. H. J., Codgell R. J. & Hunter, C. N. (1995). Heterologous expression of genes encoding bacterial light-harvesting complexes in Rhodobacter sphaeroides. J.
Biol. Chem. 270, 23875-23882.
Girvin, M. E., Rastogi V. K., Abildgaard F., Markley, J. L. & Fillingame, R.
H.
(1998). Solution structure of the tansmembrane H+ - transporting subunit c of the F~Fo.
ATP synthase. Biochemistry 37, 8817-8824.
Glazebrook, T. S., Cannon, G. C. & Shiveley, J.M. (1979). Intracytoplasmic membrane production in Escherichia coli O1 11a: Isolation and lipid characterization of cell membranes Current microbiology. 2, 5-1 0.
Gong, F. C., Giddings, T. H., Meel J: B., Staehelin, L. A. & Galbraith, D. W.
(1996).
Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins. Proc. Natl. Acad. Sci. USA. 93, 2219-2223.
Hiraoka S., Matsuzaki H. & Shibuya I. (1993). Active increase in cardiolipin synthesis in the stationary growth phase and its physiological significance in Escherichia coli FEBS Letters 336, 221-224.
Hockney, R. C. (1994). Recent developments in heterologous protein production in Escherichia coli. Trends Biotechnol. 12, 456-463.

Ingraham, J. L.. Maaloe, O. & Neidhart F. C. (1983). Growth of the bacterial cell.
Sinauer Associates. Sunderland. Mass.
Kellenberger E. & Ryter A. (1958). Cell wall and cytoplasmic membrane of E.
coli.
J. Biophys Biochem. Cytol. 4, 323-326.
Kurland C. G. & Dong H. (1996). Bacterial growth inhibition by overproduction of protein. Molec.. Microbiol. 21, 1-4.
Laemli, U. K. ( 1970). 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 modulate peptide insertion into membranes Biochemistry 36, 5476-5482.
1~
Miroux B. & Walker J. E. (1996). Over-production of proteins in Escherichia coli:
mutant hosts that allow synthesis of some membrane proteins and globular proteins at 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). Proliferation of intracellular membrane structures upon homologous overproduction of cytochrome P-450 in Candida maltosa. Biochim.
Biophys. Acta. 1236, 163-169.
Osborn, M. J., Gander, J. E., Parise, E. & Carson, J. (1972). Mechanism of assembly of the outer membrance of Salmonella Typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J. Biol. Chem. 247, 3962-3972.
Perlin, D. S., Cox; D. N. & Senior A. E. (1983). Integration of F, and the membrane sector of the proton-ATPase of Escherichia coli. Role of subunit b (uncF
protein) J.
Biol. Chem. 258,9793-9800.

Raetz, C. R. H. (1978). Enzymology, genetics, and regulation of membrane phospholipids synthesis in Esclierichia coli. Microbiol. Rev. 42; 614-659.
5 Randle C. L., Albro P. N. & Dittmer J. C. (1969). The phosphoglyceride composition of Gram-negative bacteria and the chances in composition during growth.
Biochim.
Blophys. Acta 187; 214-220.
Rouser, G., Fleischer, S. & Yamamoto, A. (1970). Two dimensional thin-layer 10 chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5, 494-496.
Schnaitman C. & Greenwalt J. W. (1966). Intracytoplasmic 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. Methods Enzymol. 185, 60-89.
Supply, P., Wach, A., Throes-Sempoux, D., & Goffeau A. (1993). Proliferation of intracellular- structures upon overexpression of the PMA2 ATPase in Saccharomyces cerevisie. J. Biol. Chem. 268, 19744-19752.
van Klompenburg, W., Nilsson, L, von Heijne, G., & de Kruijff B. (1997).
Anionic phospholipids are determinant of protein topology. EMBO J. 16, 4261-4266.
2j von Meyenburg, K., Jorgensen B.B. & Reurs B. (1984). Physiological and morphological effect of overproduction of membrane-bound ATP synthase in Escherichia coli K-12. EMBD J., 3; 1791-1797.

Walker, J. E., Saraste, M., & Gay, N. J. (1982). E. coli F~-ATP synthase interacts with a membrane protein component of a proton channel. Nature 298, 867-869.
Walker, J. E., Saraste, M., & Gay, N. J. (1984). The unc operon. Nucleotide sequence, regulation and structure of ATP synthase. Biochim. Biophys. Acta 768, 164-200.
Walker, J. E. & Saraste, M. (1996) Membrane protein structure. Editorial overview.
Curr. Op. Struct. Biol. 6, 457-459.
Weigand R. A., Shiveley J. M. & Greenwalt J. W. (1970). Formation and ultrastructure of extramembranes in Escherichia coli. J. Bacteriol. 102, 240-249.
Weiner J. H., Lemire B. D., Elmes M. L., Bradley R. D. & Scraba D. G. (1984) Overproduction of fumarate reductase in Escherichia coli 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). Crystalline arrays of the Escherichia coli sn - glycerol - 3 - phosphate acyltransferase, an integral membrane protein. J. Biol. Chem. 261; 9951-9958.
Yamato L, Futai M., Anraku Y. & Nonomura Y. (1978). Cytoplasmic membrane vesicles of Escherichia coli. J.Biochem. 83, 117-128.

Sequence Listing SEQ ID NO: 1 forward primer E. coli unc E l unc F genes - Example 1 TAG GAA TTC ATA TGG AAA ACC TGA ATA TGG ATC TGC TGT
SEQ ID NO: 2 reverse primer E. coli unc E l unc F genes - Example 1 CGA AAG CTT TTA TTA CAG TTC AGC GAC AAG TTT ATC CAC G
SEQ ID NO: 3 intracistronic sequence - artificial sequence - Example 1 AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA
SEQ ID NO: 4 forward primer - b subunit - Example 1 TAG GAA TTC ATA TGA ATC TTA ACG CAA CAA TCC TCG GCC
SEQ ID NO: S
reverse primer - b subunit - Example 1 ATC TCC TTC TTA AAG TTA AAC AAA ATT ATT TTA CAG TTC AGC GAC
AAG TTT ATC
SEQ ID NO: 6 c subunit - forward primer - Example 1 TTT GTT TAA CTT TAA GAA GGA GAT ATA ATG GCT TCA GAA AAT ATG
ACG CCG
SEQ ID NO: 7 c subunit - reverse primer - Example 1 ,0 CGA AAG CTT TTA CTA CGC GAC AGC GAA CAT CAC GTA CAG

SEQ ID NO: 8 N-terminal sequence of trypsinised polypeptides comprising b subunit - see Example 4 SQILDEKAE

Claims (26)

Claims

1. A method for producing a polypeptide, comprising the steps of:

a) providing a bacterial host cell comprising an intracytoplasmic membrane, wherein the intracytoplasmic membrane has a composition which differs from other membranes in the host cell;

b) transforming the host cell with a nucleic acid construct comprising a coding sequence encoding the polypeptide and expressing the polypeptide in the bacterial host cell such that it becomes associated with the intracytoplasmic membrane;
and c) isolating the polypeptide by separating the intracytoplasmic membrane from other cellular components.

2. A method according to claim 1, wherein intracytoplasmic membrane production is induced in the bacterial host cell prior to, or simultaneously with, the expression of the polypeptide.

3. A method according to claim 1 or claim 2, wherein the intracytoplasmic membrane differs from other membranes in the host cell by having one or more properties selected from the group consisting of:

a) a lipid:protein ratio of greater than about 0.4 b) a membrane phospholipid composition comprising greater than about 4%
cardiolipin;

c) a membrane phospholipid composition comprising less than about 20%
phosphatidyl glycerol; and d) a membrane phospholipid composition comprising lower levels of phosphatidylethanolamine.

4. A method according to claim 2 or claim 3, wherein intracytoplasmic membrane production is induced by expression, in the host cell, of the b and/or c subunit of ATP synthase, or a fragment thereof capable of inducing membrane proliferation.

5. A method according to claim 4, wherein the ATP synthase is the Fo membrane sector of E. coli ATP synthase.

6. A method according to claim 4 or claim 5, wherein the fragment of ATP
synthase consists essentially of amino acids 1-25, 1-34, 1-48, 1-157 or 25-157 of subunit b of ATP synthase.

7. A method according to any one of claims 4 to 6, wherein the host cell is transformed with a nucleic acid construct comprising a coding sequence encoding the b and/or c subunit of ATP synthase, or a fragment thereof capable of inducing membrane proliferation, operatively linked to a promoter.

8. A method according to claim 7, wherein the b and/or c subunit of ATP
synthase, or a fragment thereof capable of inducing membrane proliferation, and the polypeptide, are encoded on the same nucleic acid construct.

9. A method according to claim 8, wherein the coding sequence encoding the b and/or c subunit of ATP synthase, or a fragment thereof capable of inducing membrane proliferation, is placed upstream of the coding sequence encoding the polypeptide and the two coding sequences are expressed in tandem from the same promoter.

10. A method according to any preceding claim, wherein the host cell is an E.
coli cell.

11. A method according to claim 10, wherein the cell is or is derived from a cell selected from the group consisting of 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).

12. A method according to any preceding claim, wherein the polypeptide is a membrane protein.

13. A method according to any preceding claim, wherein the coding sequences encoding the polypeptide and/or the subunit b and/or c of ATP synthase are operatively linked to an inducible promoter.

14. A nucleic acid construct for the expression of a polypeptide in a bacterial host cell, comprising a coding sequence encoding the polypeptide and a coding sequence encoding a b and/or c subunit of ATP synthase, or a fragment thereof capable of inducing membrane proliferation.

15. A nucleic acid construct according to claim 14, wherein the coding sequence encoding the b and/or c subunit of ATP synthase, or a fragment thereof capable of inducing membrane proliferation is placed upstream of the coding sequence encoding the polypeptide.

16. A nucleic acid construct according to claim 14 or claim 15, wherein the coding sequence encoding the b and/or c subunit of ATP synthase, or a fragment thereof capable of inducing membrane proliferation, and/or the coding sequence encoding the polypeptide is under the control of an inducible promoter.

17. A nucleic acid construct according to claim 16, wherein the coding sequence encoding the b and/or c subunit of ATP synthase, or a fragment thereof capable of inducing membrane proliferation, and the coding sequence encoding the polypeptide are under the control of the same promoter.

18. A nucleic acid construct according to any one of claims 14 to 17, which further comprises a coding sequence encoding a detectable label.

19. A nucleic acid construct according to claim 18, wherein the detectable label is green fluorescent protein.

20. A 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 which is recognised by a bacteriophage RNA polymerase.

21. A nucleic acid construct according to any one of claims 14 to 20, wherein the ATP
synthase is the Fo membrane sector of E. coli ATP synthase.

22. A nucleic acid construct according to any one of claims 14 to 21, wherein the fragment of ATP synthase consists essentially of amino acids 1-25, 1-34, 1-48, 157 or 25-157 of subunit b of ATP synthase.

23. A host cell comprising a nucleic acid construct according to any one of claims 14 to 33.

24. A host cell according to claim 23 which is an E. coli cell as described in claim 10 or claim 11.

25. A method of screening agents which bind to, affect or modulate a desired membrane protein, comprising the steps of transforming the host cell with a vector according to any one of claims 14 to 24; inducing expression of the desired membrane protein;
culturing the host cells to produce the desired membrane protein; immobilising cell membranes on a support and exposing the membranes to the agent to be screened under conditions which promote the interaction of the agent with the polypeptide.

26. A method of screening agents which bind to, affect or modulate a desired polypeptide, comprising the steps of:

(a) transforming a host cell with a vector according to any one of claims 14 to 24;

(b) inducing expression of ATP synthase subunit b or subunit c from the first expression unit, and culturing the host cells such that membrane production is induced:

(c) inducing expression of the desired membrane protein from the second expression unit and culturing the host cells to produce the desired membrane protein; and (d) immobilising the cells on a support and exposing the cells to the agent to be screened under conditions which promote the interaction of the agent with the polypeptide.