WO2009050498A2

WO2009050498A2 - Chimeric constructs for eukaryotic membrane expression in prokaryotic cells

Info

Publication number: WO2009050498A2
Application number: PCT/GB2008/003573
Authority: WO
Inventors: Bonnie Ann Wallace
Original assignee: BIRKBECK
Current assignee: BIRKBECK
Priority date: 2007-10-19
Filing date: 2008-10-20
Publication date: 2009-04-23
Anticipated expiration: 2010-04-19
Also published as: WO2009050498A3; GB0720563D0

Abstract

An isolated nucleic acid molecule encoding a chimeric membrane protein is provided. The chimeric membrane protein comprises at least two N-terminal transmembrane segments of a prokaryotic membrane protein and the remaining transmembrane segments are eukaryotic. A chimeric membrane protein is also provided. The nucleic acid molecule encoding the chimeric membrane protein can be inserted into a prokaryote, for example, a bacterium such as E. coli. This can be done by introducing a vector into the prokaryote. The chimeric membrane protein encoded by the nucleic acid molecule is then expressed in the prokaryote.

Description

Protein

Field of the Invention

The invention relates to constructs for producing membrane proteins, especially eukaryotic membrane proteins, using bacteria, and to methods of producing such proteins.

Background

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.

Summary of the Invention

The inventors have developed a chimeric expression system, combining portions from prokaryotic membrane proteins with portions of the desired eukaryotic membrane protein and have enabled expression in prokaryotes. Accordingly, there is provided, 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. Without being bound by a particular theory, it is the inventor's belief that 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. In order to attain correct folding of the membrane protein and functionality, it is preferred that the prokaryotic membrane protein is an homologue of the eukaryotic membrane protein. Specifically, it is preferred that 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.

Further, it is preferred that 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 α-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. In particular, 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. Alternatively, the segments may also be artificially created segments or modified segments. For example, 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. In some cases, 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. It may be useful to alter the structure of one or more segments of a transmembrane protein to investigate the functionality of that protein and the individual segments. 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. There is no requirement for the nucleotide sequence encoding a particular segment to 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. Where a naturally occurring protein is used though, it is preferable that 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. In particular, the protein may comprise an additional amino acid sequence from either the eukaryote or prokaryote from which the transmembrane segments are taken. Alternatively, 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. In particular, 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.

In one embodiment of the invention, 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.

Also provided is an isolated membrane protein encoded by the nucleic acid of the invention or a functional fragment thereof. The isolated membrane protein 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).

Further provided is 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. Preferably the expression vector is an E.coli vector.

Preferably 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. It is further preferred that the vector comprises any other regulatory sequences required to obtain expression of the nucleic acid molecule. For example, the vector may also include plasmids to further aid expression such as rosetta (codon plus) or PLysS which suppresses the promotor at other sites.

Also provided is a 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.

Preferably 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.

The invention will now be described in detail by way of example only, with reference to the drawings in which:-

Brief description of the drawings

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.), b) Western blot of whole cells for all six chimeric constructs isolated from 22 degree autoinduction growths in C41(D3), c) Western blot of whole cells for all six chimeric constructs isolated from 37 degree autoinduction growths in C41(D3).

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.

Detailed Description of the Invention Examples Example 1 Production of a first chimeric protein according to the invention.

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. In this case 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.

Using bioinformatics 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. We then constructed the N-terminal prokaryote/C-terminal eukaryote, in which the surrounding transmembrane helices that interact with the membrane and are essential for folding and membrane insertion are prokaryote, but the core transmembrane helices that form the pore (in the case of the channels) are from the eukaryote. 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.

Two constructs were made using standard techniques. The first construct contained segments 1 to 4 with the loop between segments 4 and 5 of the prokaryote and segments

5 and 6 from the eukaryote. 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.

One example of 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.

Example 2

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

To show that the technology is suitable for use with more than one type of eukaryotic partner, 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.

Example 4

Constructs according to the invention can be made using splices in different positions. In other words, 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. This is potentially important for drug development as the S4-S5 linker has been shown to have an important role in the channel functional properties, so these constructs would allow testing of the role of that region in drug binding. Another example of this is the NavBac3.2 and NavBac3.4 pairing.

Example 5

As mentioned above, the constructs may comprise additional, non-transmembrane regions. To show that the technology is suitable for use with constructs that included additional domains (for example, the C-termini) of more than one partner pairs of constructs including additional sequences were created. 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. In this case, 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 6

The six constructs were expressed in both cell lines C41(DE3) and C41(DE3)pLysS to show that growth was not limited to a single cell line (Figure 8a and Figure 8b). Although the growth rates differed slightly between the different constructs, and were generally (although not always) lower with respect to the growth rates for the parent bacterial NaChBac construct, none of the constructs killed the cells in either cell line and all produced reasonable growth in at least one of the cell lines.

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. In addition, the constructs in C41 (DE3) grown under both IPTG-induction and auto induction conditions produced protein (Figures 7a and 7b, respectively) showing that the expression was possible using more than one type of induction procedure. Growth conditions could be selected by the skilled person to suit the construct and cell line used.

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.

In conclusion, the inventor has shown that 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.

Claims

1. An isolated nucleic acid molecule encoding a chimeric membrane protein, comprising at least two N-terminal transmembrane segments of a prokaryotic membrane protein, the remaining transmembrane segments being eukaryotic.

2. A nucleic acid molecule according to claim 1, wherein the membrane protein is an ion channel or G-protein coupled receptor.

3. A nucleic acid molecule according to claim 1 or claim 2, comprising at least three prokaryotic membrane spanning segments.

4. A nucleic acid molecule according to any preceding claim, comprising a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in figure 1 or 6.

5. A nucleic acid molecule according to claim 4, comprising a nuceotide sequence consisting of any of the the nucleotide sequences shown in figure 1 and figure 6.

6. An isolated membrane protein encoded by the nucleic acid of any preceding claims.

7. An isolated membrane protein according to claim 6, wherein the protein comprises the amino acid sequence in figure 1 or 6.

8. A membrane protein according to claim 6, further comprising additional amino acids.

9. A vector comprising the nucleic acid of any of claims 1 to 5.

10. A prokaryotic host cell comprising the vector of claim 8.

11. A host cell according to claim 9, wherein the host cell is from the species E. coli.

12. A method for producing a membrane protein, comprising expressing a protein encoded by the nucleic acid of any of claims 1 to 5 in a prokaryote.

13. The method of claim 11, further comprising the step of purifying the protein.