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WO2009050498A2 - Protéine - Google Patents

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Publication number
WO2009050498A2
WO2009050498A2 PCT/GB2008/003573 GB2008003573W WO2009050498A2 WO 2009050498 A2 WO2009050498 A2 WO 2009050498A2 GB 2008003573 W GB2008003573 W GB 2008003573W WO 2009050498 A2 WO2009050498 A2 WO 2009050498A2
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WO
WIPO (PCT)
Prior art keywords
protein
nucleic acid
membrane protein
eukaryotic
prokaryotic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2008/003573
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English (en)
Other versions
WO2009050498A3 (fr
Inventor
Bonnie Ann Wallace
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BIRKBECK
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BIRKBECK
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Filing date
Publication date
Application filed by BIRKBECK filed Critical BIRKBECK
Publication of WO2009050498A2 publication Critical patent/WO2009050498A2/fr
Publication of WO2009050498A3 publication Critical patent/WO2009050498A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the invention relates to constructs for producing membrane proteins, especially eukaryotic membrane proteins, using bacteria, and to methods of producing such proteins.
  • Membrane proteins are important and significant targets of a significant proportion of current pharmaceutical drugs. Expression of eukaryotic membrane proteins in eukaryotic systems generally produces only a limited amount of protein and expression in prokaryotic systems has proved difficult. However, it is possible to express heterologous prokaryotic membrane proteins in prokaryotes. It would be advantageous to be able to utilise the expression volumes attainable using prokaryotic systems, especially E. coli, in order to produce larger volumes of eukaryotic membrane proteins.
  • an isolated nucleic acid encoding a chimeric membrane protein, the protein comprising at least two N-terminal transmembrane segments of a prokaryotic membrane protein, the remaining transmembrane domains being eukaryotic.
  • the N-terminal portion of the eukaryotic protein is replaced by the corresponding portion of a homologous prokaryotic protein.
  • the prokaryotic portions are recognised by the translation and folding machinery of the expression prokaryote and that the prokaryote is then tricked into continuing expressing the protein when it reaches the foreign eukaryotic portions.
  • the prokaryotic membrane protein is an homologue of the eukaryotic membrane protein.
  • the amino acid sequence of the prokaryotic membrane protein domains has at least 20% sequence identity with the eukaryotic domains replaced. More preferably, the sequences are at least 22% identical, more preferably at least 25% identical, even more preferably at least 27% identical.
  • the corresponding amino acid sequences in the chimeric protein have at least 70% homology, more preferably at least 80%, even more preferably at least 90% homology with the relevant sequences of either the eukaryotic protein or prokaryotic protein from which the transmembrane segments are taken.
  • Membrane proteins are proteins found in the cell membrane and include proteins such as ion channels and cell surface receptors. It is preferred that the membrane proteins is an ion channel or a G-protein coupled receptor (GPCR). In particular, when the membrane protein is an ion channel, it may be, for example, a sodium channel, a calcium channel or a potassium channel. As is well known in the art, such proteins are made up of a series of membrane spanning (transmembrane) segments, particularly ⁇ -helices, connected by intra- and extra-cellular loops.
  • the chimeric membrane protein comprises at least two prokaryotic membrane spanning segments, but preferably comprises at least three, or more preferably at least four prokaryotic transmembrane segments.
  • any segments required for folding or membrane insertion are preferably prokaryotic.
  • the chimeric membrane protein also preferably comprises the eukaryotic segments essential for function. In particular, where the membrane protein contains transduction or core regions, these are preferably eukaryotic. Where the chimeric membrane protein comprises more than two prokaryotic membrane spanning segments, the two segments at the N-terminal domain are preferably completely prokaryotic, but later domains may be part prokaryotic and part eukaryotic. Alternatively, each membrane spanning segment may be entirely prokaryotic or eukaryotic, and the prokaryotic and eukaryotic segments joined at a loop.
  • the joining loop may be intracellular or extracellular and may be taken from the prokaryote or the eukaryote or may be a combination of the two.
  • the splice point, or point at which the eukaryotic and prokaryotic regions are joined, may be selected according to the intended use of the protein. For example, where a particular eukaryotic segment or joining loop is thought to be important for function, for example for binding a ligand or drug, or in the case of ion channels, voltage sensing and/or inactivation, two constructs may be prepared, one in which that segment or joining loop is eukaryotic and one in which the segment or joining loop is prokaryotic. The activity of the two proteins can then be compared to assess the importance of the segment or region.
  • the transmembrane segments are preferably naturally occurring transmembrane segments, having naturally occurring amino acid sequences.
  • the segments may also be artificially created segments or modified segments.
  • the amino acid sequences of naturally occurring transmembrane segments may be modified by making substitutions, replacing one or more amino acids, or deleting or adding one or more amino acids.
  • the modifications are conservative, for example, replacing one amino acid with a similar amino acid, but in other cases, the modifications are non-conservative. In those cases, it is preferable, that any changes made do not affect the protein being able to fold and function as the naturally occurring protein. Non- conservative modifications may be used, for example, to modify protein folding or function.
  • Modified proteins preferably have an amino acid sequence having at least 70%, more preferably at least 80%, even more preferably at least 90% homology to the amino acid sequence of the naturally occurring protein.
  • the nucleic acid of the invention encodes the amino acid sequence of the desired protein.
  • the nucleotide sequence encoding a particular segment may be the same as the naturally occurring nucleotide sequence encoding that segment.
  • the nucleotide sequences may be artificial or modified, for example, to improve codon usage.
  • nucleotide sequence is at least 70% homologous to the naturally occurring nucleotide sequence encoding the relevant segments of that protein.
  • the protein may also comprise amino acids in addition to the transmembrane segments.
  • the protein may comprise an additional amino acid sequence from either the eukaryote or prokaryote from which the transmembrane segments are taken.
  • the additional amino acid sequence may be from a different organism or may be an artificial sequence.
  • the additional amino acid sequence may be from a transmembrane segment, but is preferably an extramembranous region.
  • the additional amino acid sequence is preferably a sequence taken from or encoding a region found in the extramembranous C-terminal regions found in one of the eukaryote or prokaryote from which the transmembrane segments are taken. Additional amino acid sequences may be useful to improve or modulate the function of the chimeric protein or enable the protein to interact with other molecules, especially other proteins.
  • the protein may be modified by the addition, for example, of a lipid or a carbohydrate. Such additions may help with, for example, anchoring the protein in the membrane.
  • Other optional additions include functional groups such as labels, especially fluorescent or similar labels which can be used, for example, to monitor protein function.
  • the nucleic acid has or comprises a sequence having at least 70% homology with one of the nucleotide sequences for chimeras shown in figure 1 and figure 6 (or SEQ ID NOs 1 and 13 to 18). More preferably it has or comprises a sequence having at least 75% homology, more preferably at least 80% homology, more preferably at least 85%, more preferably at least 90% homology with the relevant sequence.
  • an isolated membrane protein encoded by the nucleic acid of the invention or a functional fragment thereof may additionally comprise a tag, such as a histidine tag, to enable its purification.
  • a functional fragment is a portion of the whole peptide encoded by the nucleic acid of the present invention that has the same or similar function.
  • the protein may comprise an amino acid sequence selected from the amino acid sequences for chimeras shown in figures 1 and 6 (SEQ ID NOs 3 and 6 to 11).
  • an expression vector comprising the nucleic acid of the invention.
  • Expression vectors are well known for enabling the introduction of nucleic acids into host cells. All such expression vectors are well known to those skilled in the art and the use of expression vectors in order to express the nucleic acid sequence is a standard technique well known to those skilled in the art.
  • the expression vector is an E.coli vector.
  • the expression vector of the present invention comprises a promoter and the nucleic acid molecule of the present invention.
  • the vector leads to the production of the protein of the present invention.
  • the vector comprises any other regulatory sequences required to obtain expression of the nucleic acid molecule.
  • the vector may also include plasmids to further aid expression such as rosetta (codon plus) or PLysS which suppresses the promotor at other sites.
  • prokaryotic host cell transformed with the vector of the invention. Any appropriate host cell may be used, such as E. coli.
  • the prokaryotic membrane spanning segments may be from any prokaryote which has a homologue to the eukaryotic protein of interest.
  • the prokaryote used as the host may be from the same or different species as the species from which the prokaryotic membrane spanning segments are taken.
  • the eukaryotic membrane spanning segments may be from any eukaryote for which a prokaryotic homologue can be identified. It is preferred that the eukaryote is a mammal, especially a primate, particularly a human.
  • the present invention further provides a method for producing the membrane protein of the present invention comprising transfecting a host cell with the vector of the present invention, culturing the transfected host cell under suitable conditions in order to lead to the expression of the nucleic acid molecule and production of the peptide of the present invention.
  • the peptide may then be harvested from the transfected cells.
  • the method also comprises the step of purifying the protein.
  • Purification steps may include affinity purification using a tag on the protein, gel filtration and optionally a centrifugation step.
  • Figure 1 shows the nucleotide and amino acid sequences of a first nucleic acid and protein according to the invention, indicating which derive from the prokaryotic and eukaryotic partners.
  • Figure 2 is a western blot of the whole cell extract and the membrane fraction of the first protein according to the invention, showing both the intact prokaryotic protein (Nachbac) and the chimera.
  • Figure 3 is a schematic diagram showing the construction of a vector according to the invention.
  • Figure 4 compares the prokaryotic and eukaryotic sequences used.
  • Figure 5 shows six additional chimeric constructs according to the invention.
  • the figure shows examples of six sodium channel chimeric constructs created using NaChBac as the bacterial partner and either Domain II ( Figure 5a) or Domain III ( Figure 5b) of human NaVl .7 as the eukaryotic partner.
  • Figure 6 provides a) Protein sequences of the six chimeric constructs diagrammed in Figure 5, showing the bacterial partner sequence (underline) and the eukaryotic partner (no underline) and the homology alignments of the bacterial partner and the chimeric proteins for each construct, b) DNA sequences of all the chimeras (confirmed by sequencing).
  • Figure 7 shows a) SDS gel and Western blot of whole cells isolated from IPTG-induced C41(D3) growths at 37 degrees of all six chimeric constructs shown in figure 6 (plus the parent NaChBac - loaded at 5x lower amount, for comparison).
  • the arrow indicates the approximate migration position of the chimeras (they all have slightly different molecular weights as indicated on the figure, depending on the number of amino acids in the construct.)
  • Figure 8 shows growth curves for cells containing the NaChBac parent and the six chimeric constructs, under different conditions: a) in C41(D3) cells, induced with 1 mM IPTG and grown at 22 degrees, b) in C41(D3) pLysS, induced with 1 mM IPTG and grown at 22 degrees, c) in C41(D3), induced with 1 mM IPTG and grown at 37 degrees, and d) in C41 (D3)pLysS, induced by 1 mM IPTG and grown at 37 degrees.
  • Figure 9 shows a) Demonstration that the chimeric protein is located in the detergent- solubilisable fraction, not in inclusion bodies, b) Preliminary purifications of two of the constructs (2 and 4) solubilised in the detergent dodecylmaltoside using a nickel affinity column to bind the N-terminal His tags.
  • Columns 2 and 3 are of construct 2, run without (column 2) and with (column 3) DTT in the running buffer;
  • Columns 3 and 4 are of construct 3, again run without and with DTT. The approximate positions of the chimeras are indicated by the arrows.
  • Figure 10 shows a) One-step affinity purification of all six chimeras.
  • the arrow indicates the position of the chimeras, b) Purification steps for chimera 2 first on a nickel-affinity column, followed by ion exchange on an SP column.
  • the chimeric protein locations are indicated by the boxes.
  • the inventors cloned a chimera of a four-transmembrane N-terminal segment from the B. halodurans sodium channel (NaChbac) plus the two-transmembrane "channel forming" segment from a eukaryotic partner.
  • the inventors used a lower order eukaryote, a jellyfish (Cyanea).
  • the whole eukaryote protein would not express in E. CoIi at any detectable levels, but the chimera did express, was solubilised in detergent and purified.
  • the inventor identified the homologues, the transmembrane boundaries and the functionally-important regions that must be eukaryotic in the chimera.
  • the two homologues had 25.5% identity.
  • the N-terminus of the prokaryotic protein is recognised by the E. coli translation and folding machinery, which is then tricked into carrying on when it reaches the "foreign" eukaryotic protein at the C-terminus, producing a folded and properly membrane-associated protein with the functional characteristics of the eukaryote.
  • the first construct contained segments 1 to 4 with the loop between segments 4 and 5 of the prokaryote and segments
  • the second construct contained the same segments, but the loop was taken from the eukaryote. Both were cloned into TOPO and sequenced. They were confirmed to have greater than 99% of the expected sequence. The first construct was then used to create the membrane protein.
  • the first construct was inserted into three E. coli cell lines under various conditions.
  • the cell lines used were C41(DE3), C43 and BL21, all of which are commercially available
  • the vectors used were pPROExHTb, pET-30a, PQE60, and pTrcHis.
  • the inventors were able to produce the protein with at least one example of each vector and one example of each cell line. The identity of the protein was confirmed because it had the correct molecular weight, was found in the membrane fraction and stained with anti-His antibodies.
  • the protein was purified using a Talon column, which uses a standard purification method based on the His tag.
  • the protein had the correct molecular weight, including the expected multimers.
  • NavBac2.1, NavBac2.2, NavBac3.1, NavBac3.2, NavBac3.3, and NavBac3.4 Six different constructs (designated NavBac2.1, NavBac2.2, NavBac3.1, NavBac3.2, NavBac3.3, and NavBac3.4) involving the bacterial partner NaChBac and two different domains of the human sodium channel NaV 1.7 were produced.
  • the constructs produced are shown in figure 5 and their protein and DNA sequences are shown in figure 6.
  • the constructs were of a type similar to that described in example 1, but used different transmembrane segments, obtained from different partners.
  • Appropriate vectors were prepared and used to insert the constructs into host cells (C41(DE3) and C41(DE3)pLysS, mutant E. coli cell lines) and the proteins expressed..
  • the proteins produced were of the correct size and were identified by Western blotting. This is shown in figure 7a.
  • the inventor attempted to express the eukaryotic proteins from which the eukaryotic segments were obtained in E. coli, but was unsuccessful using a wide range of conditions, including those used to produce the chimeras.
  • Example 3 The inventor attempted to express the eukaryotic proteins from which the eukaryotic segments were obtained in E. coli, but was unsuccessful using a wide range of conditions, including those used to produce the chimeras.
  • pairs of constructs having the same splice point but including different eukaryotic sequences were prepared.
  • Such pairs include NavBac2.1 and NavBac3.1, which includes two different domains from NaVl .7, and NavBac2.2 and NavBac3.2.
  • Constructs according to the invention can be made using splices in different positions.
  • different constructs may be prepared using the same partners, but linked at different splice points.
  • the NavBac3.1 and NavBac3.3 pair were created to show that using the same bacterial and eukaryotic partners, constructs with different splice points could be produced. In this case, one had a splice point before the S4-S5 linker and the other had the splice point after the linker. This created, respectively, a construct with the linker from the eukaryotic partner and a construct with the linker from the bacterial partner.
  • the constructs may comprise additional, non-transmembrane regions.
  • additional domains for example, the C-termini
  • the NavBac2.1 and NavBac2.2 pair were created to show that the chimeras could also include additional sequences beyond the transmembrane regions from either partner.
  • constructs with the extramembranous C-terminal regions from either the bacterial or eukaryotic partner were expressed.
  • the NavBac3.1 and NavBac3.2, and the NavBac3.3 and NacBac3.4 pairs were similar examples.
  • Example 7 The constructs in both the C41 (DE3) and C41(DE3)pLysS cell lines were grown at both 22 degrees and 37 degrees ( Figures 8a to 8d) to show that the growth was possible under more than one growth condition. There was a difference in growth rates at the two temperatures, as expected, but cells did grow at both temperatures for all six constructs.
  • Example 8 The constructs may be produced using different vectors. The constructs were produced using both pCold and pET28a vectors. Both vectors were successful for all constructs except construct 2 in pCold.
  • Example 9 In addition to demonstrating that the proteins were expressed, studies were done to show that the proteins were present in the detergent-solubilisable fraction (i.e. membranes), as opposed to in inclusion bodies (Figure 5a). Also, initial purification studies of two of the constructs using nickel affinity were carried out, showing they have the correct molecular weight. Also, the constructs were identified on Western blots ( Figure 9b). Finally, preliminary purifications of all six constructs were done using a batch affinity method ( Figure 1 Oa) and a two-step purification of chimera 2 was done ( Figure 1 Ob), and the product identified by Western blotting.
  • constructs according to the invention may be created using a variety of eukaryotic and prokaryotic partners, including chimeras with human eukaryotic partners, and that growth and expression can be achieved in more than one cell line using more than one vector, under more than one induction condition and using more than one growth condition.

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Abstract

La présente invention concerne une molécule d'acide nucléique isolée codant pour une protéine membranaire chimère. La protéine membranaire chimère comprend au moins deux segments transmembranaires N-terminaux d'une protéine membranaire procaryote, tandis que les segments transmembranaires restants sont eucaryotes. L'invention concerne également une protéine membranaire chimère. La molécule d'acide nucléique codant pour la protéine membranaire chimère peut être insérée dans un organisme procaryote, par exemple dans une bactérie comme E. coli. Cela peut être fait en introduisant un vecteur dans l'organisme procaryote. La protéine membranaire chimère encodée par la molécule d'acide nucléique peut ensuite s'exprimer dans l'organisme procaryote.
PCT/GB2008/003573 2007-10-19 2008-10-20 Protéine Ceased WO2009050498A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0720563.6 2007-10-19
GB0720563A GB0720563D0 (en) 2007-10-19 2007-10-19 Protein

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WO2009050498A2 true WO2009050498A2 (fr) 2009-04-23
WO2009050498A3 WO2009050498A3 (fr) 2009-06-04

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010073009A3 (fr) * 2008-12-22 2010-09-10 Universitetet I Oslo Composés, compositions et utilisation

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040323A2 (fr) * 2001-11-08 2003-05-15 Children's Medical Center Corporation Canal ionique bacterien et technique de recherche de modulateurs du canal ionique
US20050202539A1 (en) * 2004-03-11 2005-09-15 Hydra Biosciences System for screening eukaryotic membrane proteins

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010073009A3 (fr) * 2008-12-22 2010-09-10 Universitetet I Oslo Composés, compositions et utilisation
US8710008B2 (en) 2008-12-22 2014-04-29 Universitetet I Oslo Compounds, compositions and use

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WO2009050498A3 (fr) 2009-06-04
GB0720563D0 (en) 2007-11-28

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