EP1224306A1 - Systeme d'expression pour proteines membranaires - Google Patents

Systeme d'expression pour proteines membranaires

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Publication number
EP1224306A1
EP1224306A1 EP00968106A EP00968106A EP1224306A1 EP 1224306 A1 EP1224306 A1 EP 1224306A1 EP 00968106 A EP00968106 A EP 00968106A EP 00968106 A EP00968106 A EP 00968106A EP 1224306 A1 EP1224306 A1 EP 1224306A1
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EP
European Patent Office
Prior art keywords
membrane
subunit
polypeptide
expression
atp synthase
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EP00968106A
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German (de)
English (en)
Inventor
John Med. Res. Council Dunn Human Nut.Unit Walker
Bruno Donat Michel Miroux
Ignacio MRC Dunn Human Nutrition Unit Arechaga
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Medical Research Council
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the invention relates to an expression system for polypeptides.
  • the invention relates to host cells for polypeptide expression which have an altered membrane composition.
  • 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).
  • mutant hosts were 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.
  • 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.
  • the invention relates to a method for preparing a polypeptide in such host cells.
  • 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 other cellular components.
  • composition of the intracytoplasmic membrane advantageously confers some degree of resistance to toxic effects of protein expression, particularly membrane protein expression.
  • 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.
  • 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.
  • composition of intracytoplasmic membranes according to the present invention 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.
  • 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 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 synthase' preferably refers to subunit b or subunit c from the F 0 membrane sector of E. coli ATP synthase (Walker et al., 1982 Nature vol.298, pp867-869). In a highly preferred embodiment, this refers to subunit b of the F 0 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.
  • 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.
  • Membrane proteins are any proteins which associate with or are capable of associating with the membrane fraction of cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • expression of subunit b or subumt 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.
  • 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.
  • the promoter(s) 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.
  • the expression system may make use of the bacteriophage T7 RNA polymerase system.
  • the invention provides a method for expressing a protein in a host cell wherein the host cell express a bacteriophage RNA polymerase and wherein the expression system comprises a promoter sequence recognised by the polymerase.
  • the polymerase is T7 RNA polymerase.
  • the invention provides a method for expressing a protein in a host cell wherein the expression system comprises the expression vector pET or pMW7.
  • 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.
  • 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.
  • 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.
  • 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 agent(s) 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.
  • agent refers to any entity which is known or suspected of associating, either reversibly or irreversibly, with a polypeptide such as a membrane protein.
  • a polypeptide such as a membrane protein.
  • Supports upon which cell membranes could be immobilised are well known in the art, and include nitrocellulose based filters, supported filters or activated or coated surfaces such as ELISA plates.
  • 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; 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.
  • 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.
  • the invention relates to a method of expressing a polypeptide in a host cell wherein said host cell(s) are cultured at 25°C.
  • protein means any polypeptide of two or more amino acid residues or more in length.
  • 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.
  • 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.
  • 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(D ⁇ 3) (ECCC B96070445), E. coli C41(DE3) (ECCC B96070444), E. coli DK8(DE3)S (NCIMB 40885) or E. coli C2014(DE3) (NCIMB 40884).
  • coli strains means that the a host cell may be genetically closely related to one or more of the strains set out herein.
  • an E. coli strain would be considered to be 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 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).
  • a nucleic acid of the invention encoding a polypeptide of interest can be incorporated into vectors for further manipulation.
  • 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 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, 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.
  • 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.
  • origins of replication or autonomously replicating 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2, TRP1, or HIS3 gene.
  • 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. pUC18 or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.
  • 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 ⁇ -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-Delgamo 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.
  • 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).
  • T7 RNA polymerase a promoter of bacteriophage
  • 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).
  • the T7 RNA polymerase is produced from the ⁇ -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.
  • 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).
  • PLEX Invitrogen, NL
  • vectors containing the trc promoters such as pTrcHisXpressTm (Invitrogen) or pTrc99 (Pharmacia Biotech, SE)
  • vectors containing the tac promoter such as pKK223-3 (Pharmacia Bio
  • 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.
  • the S. pombe nmt 1 gene or a promoter from the TATA binding protein (TBP) gene can be used.
  • TBP TATA binding protein
  • hybrid promoters comprising upstream activation sequences (UAS) 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 PHO5 promoter is e.g.
  • PH05 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.
  • UAS upstream regulatory elements
  • 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.
  • 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 rnRNA, 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 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.
  • polypeptides expressed in host cells according to the invention are more likely to remain soluble than would otherwise be the case.
  • 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.
  • the culture is carried out at a reduced temperature.
  • 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 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 protein expression.
  • 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 difference(s) may be in the chemical composition of the lipid fraction, or the difference(s) may be in the ratios of the various lipid-containing components to one another, or the difference(s) 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.
  • 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.
  • 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.
  • 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.
  • subunit b or subunit c from the F 0 membrane sector of E. coli ATP synthase are useful in the preparation of host cells according to the invention.
  • subunit b of the F 0 membrane sector of E. coli ATP synthase is used in preparing host cells according to the invention.
  • 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.
  • Part(s) 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.
  • parts of subunit c which are useful in the present invention include but are not limited to the entire subunit c, or fragment(s) thereof.
  • 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.
  • 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.
  • the polypeptide may be any polypeptide for which it is desired to identify an interaction with the agents to be screened.
  • 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.
  • 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.
  • 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.
  • 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(D ⁇ 3).
  • 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 ⁇ l 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.bc ⁇ (be) and pEe.cb5 (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 4-5h, 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.
  • E. coli F,F 0 -ATPase ATP synthase
  • 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 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 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 (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).
  • FIG. 5 shows Coomassie stained polypeptides indicating protein contents of altered 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 ⁇ g 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 Hspl ⁇ 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 ⁇ m).
  • Figure 7 shows Coomassie stained polypeptides resulting from trypsinolysis of subunit b of E. coli ATP synthase incorporated into altered membranes.
  • the supematants (panel A) and pellets (panel B), respectively, obtained by trypsin digestion for the periods indicated, are analysed by SDS-PAG ⁇ .
  • Bands with M r 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 83 onwards.
  • the band indicated by an arrow with M r of 7 kDa corresponds to residues 1-36 of the subunit b.
  • EXAMPLE 1 OVER-EXPRESSION OF E. COLI ATP SYNTHASE B AND C SUBUNITS IN HOST CELLS
  • subunit b and c of ATP synthase are expressed in E. coli host strains C41(DE3) and C43(DE3).
  • 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 pMW172 (Way et al, 1990), giving rise to expression plasmids pMW172(Ecc) and pMW172(Ecb), respectively.
  • the insert of E. coli cb is generated by PCR using the forward primer
  • the construct for E. coli be has the intracistronic sequence
  • Reaction 1 for the b subunit, is carried out with the forward primer
  • Reaction 2 for the c subunit, is performed using the forward primer
  • Vectors for co-expression of subunits b and c in pMWI72 are made with subunit b promoter proximal (pEc.bc ⁇ ), and with subunit c promoter proximal (pEc.cb4 and pEc.cb5, respectively.
  • Plasmid pEc.cb4 contains the natural intercistronic sequence (Walker et al, 1984), whereas in pEc.cb5 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 NaCl, pH 7.4) to an optical density of 0.6 at 600 nm. 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).
  • 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.
  • 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 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.
  • subunit b At 25°C, the onset of production of subunit b is delayed by 2 h relative to 37°C, but 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).
  • the over-expression in C41(DE3) appears toxic, but less severe than the toxicity 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 OD 600 of 4.7, and contains twice as much (50 mg/1 of culture) of subunit c as the culture grown at 37°C.
  • over-expression of subunit c in C43(DE3) at 25°C is delayed with respect to overexpression in C41(DE3).
  • the culture reaches a final OD 600 of about 6.3, and contains a slightly lower amount of the recombinant protein (about 30 mg / 1 of culture).
  • subunits b and c 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.bc ⁇ ), 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.cb5) improves the expression of both proteins (Fig. 2).
  • 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.
  • 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).
  • bacteria are disrupted either in a French press or by EDTA- lysozyme treatment and osmotic shock (Osborn et al, 1972).
  • Supematants 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.
  • C41(DE3) 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 extensive in C43(DE3) than in C41 (DE3) (compare panels A and C in Fig. 3).
  • 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 (Osbom 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 centrifiiged directly at 10,000 x g.
  • the remaining supematants 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 centrifiiged for 18 h at 155,000 x g.
  • 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%o 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.bc ⁇ . In cells overexpressing subunit c only, no low speed pellet is obtained, and much of the overproduced subunit c appears to be in the highspeed 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 (Osbom 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.
  • 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
  • the ratio has a normal value (see Table 1), but in C43(DE3) cells over-expressing subunit b the 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. Table 1. Phospholipid contents of proliferated membranes accompanying over- expression of ATP synthase subunit b in E. coli C43(DE3).
  • membrane phospholipid analysis is carried out.
  • Lipids are extracted according to Bligh and Dyer (1959). To a portion of the membrane fractions, IM HCl (20 ⁇ l) is added with water to a total volume of 200 ⁇ l. Chloroform : methanol (1 : 2.2, v:v; 640 ⁇ l) is added and the sample is vortexed for 1 minute. Then 0.1 M HCl (200 ⁇ l) and chloroform (200 ⁇ l) are added and the sample is again vortexed for 1 minute. The mixture is centrifiiged, and the chloroform phase is removed. Chloroform (200 ⁇ l) is added and mixed with the aqueous phase for 1 minute and then the mixture is centrifiiged.
  • the chloroform fractions are pooled and mixed with buffer (50 mM Tris-HCl, pH 8.25, 0.1 M NaCl, 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 ⁇ l) 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 are analysed by thin layer chromatography (TLC) on silica 60 plates using chloroform : methanol: acetic acid, 65:25:10 (by vol.) as solvent.
  • TLC thin layer chromatography
  • 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.
  • 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 (IV) oxide) and then heating until a blue colour appeared.
  • phosphorus reagent molybdenum (IV) oxide
  • 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(D ⁇ 3) 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 in assembly of membrane proteins (Bogdanov et al, 1996).
  • 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.
  • 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.
  • 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).
  • the extrinsic domain of subunit b can be proteolysed, thereby preventing reassociation of F, (Perlin et al, 1983).
  • 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 C 8 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 5 minutes, four major proteolytic fragments with apparent M r 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 M r of 7 kDa.
  • 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.
  • C41(DE3) inclusion bodies are observed in electron micrographs of cross-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 (see Table 2).
  • 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.
  • Escherichia 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.

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Abstract

L'invention concerne un procédé de préparation d'un polypeptide, qui comporte les étapes consistant à : exprimer le polypeptide dans une cellule hôte s'utilisant dans l'expression de protéines, cette cellule hôte présentant une composition de membrane modifiée ; isoler la fraction de membrane de ladite cellule hôte ; et isoler ledit polypeptide de cette fraction de membrane.
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