WO1991009952A1

WO1991009952A1 - Lipoprotein signal peptide fused to antigenic polypeptides

Info

Publication number: WO1991009952A1
Application number: PCT/CA1990/000460
Authority: WO
Inventors: Peter Lau; Clément Rioux
Original assignee: Majesty (Her) In Right Of Canada As Represented By The National Research Council Of Canada
Priority date: 1989-12-26
Filing date: 1990-12-27
Publication date: 1991-07-11
Also published as: EP0510018A1; CA2032914A1; AU7034691A

Abstract

The present invention relates to a fusion plasmid for in vivo synthesis of a lipid modified polypeptide. The plasmid comprises a first DNA sequence encoding a lipoprotein signal peptide, preferably a bacterial lipoprotein signal peptide, and at least the first amino acid of a mature lipoprotein, preferably cysteine. In the DNA sequence, the preferred cysteine codon may be followed by codons for a few amino acids forming a β-turn structure. These amino acids may also include a specific exogenous protease recognition cleavage site. A second DNA sequence encoding the desired polypeptide can be inserted into the fusion plasmid to produce the desired lipid modified polypeptide.

Description

TITLE OF THE INVENTION

Lipoprotein signal peptide fused to antigenic polypeptides

FIELD OF THE INVENTION

The present invention relates to a fusion plasmid suitable for the expression of polypeptides of any composition or size modified in vivo by the addition of a lipid moiety. BACKGROUND OF THE INVENTION

The chemical synthesis of peptides is known to be substantially sequence dependent. The repeated presence of certain amino acid residues such as tryptophan, histidine, methionine or cysteine makes synthesis of even small peptides (i.e. less than twenty amino acids) difficult. In general, the chemical synthesis of peptides with more than forty amino acid residues is difficult.

The bacterial expression of peptides from DNA sequences carried on multicopy plasmids does not suffer from the same constraints on amino acid composition and size of the peptide products and results in a lower cost of synthesis. However, peptides synthesized in vivo are easily degrad d by the proteases contained in the transformed host cells. In an attempt to alleviate this problem, the working concept of tandem gene repetition described by Shen in (1984) Proc. Natl. Acad. Sci. 81, 4627-4631, allowed the stabilization of a specific gene product through the introduction of multiple copies of the foreign gene to be expressed. Unfortunately, apart from the fact that a repeated DNA sequence may be a target for deletion, this method requires extensive manipulations to obtain the proper gene configuration.

The synthesis of peptide fragments of proteins as well as subsequent immunization using these fragments allows production of specific anti-peptide antibodies useful for the im unological identification of particular clones from a recombinant DNA expression library. The capacity of many anti-peptide antibodies to react with sequences of the native protein is valuable in studies of protein structure and function. This property can also lead to the development of synthetic peptide vaccines.

However, short peptides cannot generally elicit production of antibodies by themselves. Thus, synthetic peptides are commonly rendered immunogenic by being coupled to a high-molecular weight carrier protein, such as keyhole limpet hae ocyanin (KLH) , which has been shown to induce a helper T-cell response.

Typically, these immunogenic peptide derivatives have been subsequently injected in the presence of adjuvants which are nonspecific stimulators of the immune response. Commonly used adjuvants are the complete (FCA) or the incomplete (FIA) Freund's adjuvants. FCA is a water-in-oil emulsion containing killed mycobacteria, whereas FIA is devoid of bacteria. The active ingredient in FIA that can substitute for the mycobacteria in FCA is muramyl dipeptide (MDP; N-acetyl- muramyl-L—alanyl-D- isoglutamine) . Although MDP has been shown to be an effective adjuvant in various systems, it is not without some undesirable effects. Hence, these immunization procedures require long time periods, multiple injections of antigens and often do not improve antibody titres significantly above the low levels obtained by injecting the free peptides.

Efforts have been made to develop adjuvants that can simultaneously perform the function of carriers. It has been demonstrated by Bessler et al. in (1985) Im unobiol, 170,239-244 and by Jung et al in (1985) Angen Chem. Int. Ed. Engl. 2.,872-873 that a specific peptide of the epidermal growth factor receptor (EGF-R amino acid number 516-529) coupled covalently to a synthetic N-terminal moiety of the lipoprotein from the outer membrane of E.coli was able to elicit an increased production of specific antibodies.

The chemical nature of the synthetic analog of the bacterial lipoprotein is N-palmitoyl-S-(2RS)-2,3- bis-(palmitoyloxy)propyl) )-cysteinyl-serine. Its abbreviation is Pam, Cys-Ser. Previously, this analog as well as the native lipoprotein of the outer membrane of E. coli, were found to be polyclonal activators for antibody production by B-lymphocytes as shown by Bessler et al. in (1985) The J. of Immunol. 135,1900-1905. In another study, Hopp demonstrated in (1984) Mol. Immunol. 2 ,13-16, that attachment to a dipal ityl-lysine moiety was able to convert small peptides of hepatitis B surface antigen into good immunogens. In both of these studies immune response was significantly enhanced in comparison to the corresponding peptide-KLH conjugate. These results suggest that conjugation of a peptide to a fatty acid carrier has an enhancing effect on the immunogenicity of the peptide concerned. Unfortunately, in both cases, the peptide and the lipid moiety are chemically synthesized and coupled ij vitro which leads to long and costly procedures. SUMMARY OF THE INVENTION In accordance with the present invention there is provided a fusion plasmid for in vivo synthesis of lipid modified polypeptides. This fusion plasmid comprises a DNA sequence encoding a lipoprotein signal peptide, and at least the first amino acid of a mature lipoprotein. Also, it may optionally comprise a short amino acid segment with a /3-turn structure or an exogenous protease recognition sequence. A DNA sequence encoding the desired polypeptide can then be inserted into the fusion plasmid downstream from the above- mentioned DNA sequence to produce a lipid modified polypeptide. The fusion plasmid of the present invention is preferably derived from a prokaryotic expression vector with signals for the strong transcription of the fusion genes and effective translation. Also, the lipoprotein signal peptide contained in the fusion plasmid will preferably be a bacterial lipoprotein signal peptide. Furthermore, the first amino acid of the mature lipoprotein will more preferably be cysteine. Immediately following the preferred cysteine codon, there may preferably be a DNA sequence coding for a short amino acid sequence with a 3-turn structure or an exogenous protease recognition cleavage sequence.

This fusion plasmid therefore allows for synthesis of polypeptides as part of recombinant lipopeptides secreted across the cytoplasmic membrane and recovered from the cells. Furthermore, the lipid moiety of the lipopeptides synthesized through the fusion plasmid of the present invention can also act as an intrinsic carrier/adjuvant for anti-peptide antibody production, making the lipopeptides useful in the development of vaccines. The presence of a hydrophobic region in the lipopeptide derivatives may facilitate their passage across biological membranes. In addition, the lipid portion can render a peptide more fat-soluble and can therefore be useful in enhancing the delivery of polypeptide drugs. Finally, the possible incorporation of specific enzymatic cleavage sites in the fusion system allows the release of free polypeptides from the lipopeptides. Also within the scope of the present invention is a recombinant fusion protein comprising a polypeptide and at least the first amino acid of a mature lipoprotein, said amino acid having attached thereto at least one fatty acid. Preferably, the first amino acid is a cysteine residue bearing fatty acids at its sulfhydryl and α-NH₂ groups. More preferably, the fusion protein will further comprise a short amino acid sequence comprising a /3-turn or the recognition sequence of an exogenous protease. IN THE DRAWINGS

Figures la and lb represent a schematic representation of the construction of the fusion plasmid pKLY3 from the commercially available prokaryotic expression vector pKK233-2.

Figure lc represents the insertion of the coding sequence for the P2 peptide of the epidermal growth factor receptor (EGF-R) into plasmid pKLY3 to produce plasmid pKLY4.

Figure 2 represents the nucleotide sequence of the Ncol-Hindlll DNA fragment inserted into plasmid pKLY2. Figure 3 represents the nucleotide sequence of the Sphl-BamHI DNA fragment inserted into plasmid pKLY3. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to fusion plasmids for the expression and in vivo lipid modification of polypeptides. The DNA sequences coding for the polypeptides are inserted in frame with a DNA region of the plasmid corresponding to the 5' end of a lipoprotein gene. Preferably, the lipoprotein amino terminus gene includes codons for the lipoprotein signal peptide, and at least the first amino acid of the mature lipoprotein, preferably a cysteine residue. Immediately following the preferred cysteine codon, there may be added codons for a short amino acid segment capable of forming a β- turn. This amino acid segment may also comprise a protease recognition cleavage site. The fusion plasmid is under the control of transcription and translation elements allowing for inducible expression in transformed bacteria. The nature of the expression vector, lipoprotein amino terminus, 0-turn peptide and polypeptide is as follows. Expression vector

The fusion plasmids can be derived from prokaryotic expression vectors replicated at a moderate or high number of copies per cell. Suitable levels of expression of the fusion genes can be obtained through the use of a strong inducible promoter giving no or very low transcription under non-induced conditions in order to prevent inhibition of bacterial growth by toxic fusion products. The presence of strong terminators of transcription downstream of the fusion gene prevents readthrough transcription that could make the plasmid unstable. The translationaϊ signals of the fusion gene consist of a ribosome binding site and an AUG initiation codon, possibly provided by the plasmid vector, which codes for the first amino acid of the lipoprotein signal peptide.

The presence in the fusion plasmid of the origin of replication of a single-stranded DNA phage allows, upon phage infection, the synthesis of high yields of single stranded DNA during replication. The single stranded DNA can be used for site directed mutagenesis on the plasmid. Lipoprotein amino terminus

Typically, the lipoprotein signal peptide to be used in the context of the present invention is a bacterial lipoprotein signal peptide having between 16 and 29 amino acids in length such as that described in Klein et al. , 1988, Protein Eng., 2:15-20.

This signal peptide will contain a positively charged amino terminal region, a central hydrophobic region and a carboxyl-ter inal region which has a recognition sequence for signal peptidase II (SPasell). The SPasell recognition sequence which is sometimes referred to as a "lipoprotein box" consists of Leu-X-Y- Cys (the position of cys is numbered +1 and leu -3). In this sequence, the cysteine is an invariant residue, leu at the -3 position can be replaced by a chemically similar residue, and X and Y are small amino acids.

Cleavage of a lipoprotein signal peptide in vivo through the action of the appropriate cell protease requires prior lipid modification of the first amino acid of the mature lipoprotein. In the case of cysteine, this lipid modification mainly consists in palmitoylation at the sulfhydryl group of the cysteine residue at position +1. In the recognition sequence of protease SPasell, the site of cleavage is on the amino terminal side of the modified cysteine of the mature protein (viz. Leu- X-Y-Cys). Thus, once the signal sequence has been cleaved, the attachment of another fatty acid to the α- NH₂ group of the cysteine residue of the mature protein, through further action of the cell's enzymes, generates a fully modified and processed lipoprotein.

Following the cysteine residue, mature lipoproteins contain a tetrapeptide such as Gln-Ala-Asn- Tyr (QANY) forming a ø-turn structure which may favor the cleavage action of SPasell. These natural tetrapeptides, which may be part of the plasmid of the present invention, can be replaced by Ile-Glu-Gly-Arg ( EGR) which is a specific recognition cleavage site for factor Xa protease as described by Nagar & Thogersen in 1984, Nature, 309:810-812. This protease cleaves on the σarboxy terminal side of the arginine residue.

It is to be understood by those skilled in the art that other protease recognition sequences such as Asp-Asp-Asp-Asp-Lys for enterokinase and Pro-X Gly- Pro-Y for collagenase (the arrows indicate the cleavage sites) also fall within the scope of the present invention. In fact, a wide variety of protease recognition sequences possessing the appropriate characteristics may be used in the context of the present invention. Polypeptide

According to the maturation process of lipoproteins, any sequence coding for a desired polypeptide, once fused to the gene portion coding for the desired lipoprotein amino terminus and preferably to those amino acids constituting a _/S-turn is expected to produce, after expression in a plasmid and in vivo processing, a polypeptide with an N-terminal lipid modified cysteine. The DNA sequence coding for the desired polypeptide will be either synthetic oligonucleotides or an appropriate restriction endonuclease fragment. The nature of the polypeptide that can be expressed in vivo is basically independent of its amino acid composition or size contrary to experience in chemical synthesis of peptides.

The lipopeptides thus synthesized can be used for many applications. Hence, they can serve to raise specific anti-peptide antibodies useful to identify particular clones in a recombinant DNA expression library. Antibodies recognizing peptide fragments in a native protein will help to study the structure and function of the protein and to develop synthetic peptide vaccines. Also, because the lipid portion of these derivatives could facilitate the passage across biological membranes, there is potential for the creation of efficient delivery systems for certain peptide drugs.

It may, in some instances, be desirable to obtain a peptide free of a lipid-modified N-terminus. In this case, the presence of a specific cleavage site for an exogenous protease at the amino terminus of the lipopeptide will allow for the release of free polypeptides through extracellular digestion of the lipoprotein with the appropriate enzyme. Process for Producing the Recombinant DNA of the Present Invention

Processes through which fusion genes are constructed are well known to those skilled in the art. In the case of the present invention, a DNA sequence coding for the desired lipoprotein amino terminus will be generally fused to the 5'-phosphate end of oligonucleotides or restriction endonuclease fragments coding for the desired polypeptide sequence. The following description of a preferred embodiment of the present invention can be used as a basis for the construction of other fusion genes and is not to be interpreted as limiting the scope of the present invention. Description of a preferred embodiment

As a model system, the gene coding for both the lipoprotein signal sequence of the ColE2- lasmid coded lysis protein, also known as bacteriocin release protein and the N-terminal 5 amino acid residues of the mature lysis protein, is fused to the synthetic oligonucleotides coding for the P2 peptide corresponding to the C-terminal phosphorylation site of EGF-R described by Hayden et, al. in (1986) Proc. West. Pharmacol. Sec. 29. 459-461 using the following procedure.

The fusion vector was constructed using plasmid pKK233-2 shown in Figure la. This plasmid derivative of pBR322, is expressed in a moderate number of copies per cell. It provides a strong regulated trp-lac fusion promoter (pTac) inducible by IPTG, an analog of lactose. Downstream of this promoter are a ribosome-binding site followed by an ATG translation initiation codon, a multiple cloning site and the rrn B transcription terminators. The origin of replication of phage Fl (obtained as a 514 bp Rsal fragment from plasmid pEMBL8) was inserted into the PvuII site of pKK233-2 to obtain pKLYl shown in Figure la.

Upon infection with M13 phage, the viral origin present on the plasmid permits phage-directed plasmid replication in a clockwise direction. This process results in the production of high yields of single stranded DNA that can be used for site directed mutagenesis. The method of Kunkel in (1985), Proc. Natl. Acad. Sci., Z2., 488-492 was used on single stranded DNA from pKLYl to eliminate the restriction sites BamHI and Sphl in between Sail and EcoRI to produce pKLY2 shown in Figure lb.

Plasmid pKLY2 was digested at the unique restriction sites Ncol and Hindlll for insertion of the synthetic oligonucleotide of Figure 2 with the N- terminal portion of the colicin E, lysis gene. Digestion at Ncol exposes the ATG start codon. After gene assembly, the ATG is fused in frame with the signal sequence of the lysis gene followed by five codons for the amino acids Cys-Gln-Ala-Asn-Tyr (CQANY) of the mature protein, a GTA codon for valine creating a unique Sna BI restriction site, a TAA stop codon and a BamHI restriction site close to the Hindlll ligation site. Tetrapeptide QANY can contribute to the formation of a β-turn. In the synthetic DNA, the cysteine codon TGT of the colicin E₂ lysis gene was replaced by TGC. This silent change introduced a unique Sphl restriction site in the recombinant plasmid pKLY3 shown in Figure lb.

Restriction digests of pKLY3 at the restriction sites Sphl and BamHI allow for directional insertion of annealed oligonucleotides or restriction fragments with sequences coding for the desired polypeptide followed by a termination codon (Method I). Alternatively, the fusion can be created by loop-in mutagenesis on pKLY3 single stranded DNA using oligonucleotides with complementary sequences for annealing (Method II). In both these methods, the nature of the coding sequences between the cysteine codon and the coding sequences of the polypeptides can be varied. The presence in the oligonucleotides of a restriction site absent on pKLY3 facilitated the selection of the utagenized plasmids. The unique restriction site SnaBI in pKLY3 allows for blunt-end ligation of restriction fragments or annealed oligonucleotides with the desired polypeptide coding capacity immediately downstream of the tyrosine residue of the mature colicin E₂ lysis protein (Method III). The fusion of the N-terminal portion of the colicin E₂ lysis protein to the peptide P2 which consists of 12 amino acids from the C-terminal phosphorylation site of the epidermal growth factor receptor (EGF-R amino acids 1137-1148) was made according to method I. The synthetic oligonucleotides contained the sequence for the tetrapeptide QANY and the peptide P2 followed by the TAA stop codon and the diagnostic restriction site Xbal as seen in Figure 3.

Ligation of the assembled oligonucleotides to plasmid pKLY3, digested with the restriction enzymes Sphl and BamHI, created plasmid pKLY4 shown in Figure lc.

The expression of the lipid-modified peptide from this plasmid was studied in the E^_ coli rec A lac I? strain DHδαF'IQ which overproduces the lac repressor active on the Tac promoter. Cells transformed with the vector (pKLY2), the fusion plasmid (pKLY3), or the recombinant plasmid (pKLY4) were grown in a defined medium containing IX M63 salts, ImM MgS0₄, 2% (w/v) casamino acids, 0.5% (v/v) glycerol, 40 μg/ml kanamycin (for maintenance of the F plasmid with the lac I gene) and 50 μg/ml ampicillin (for selection of the pKLY plasmids with the bla gene). The cells were incubated at 37°C with agitation (250 rpm). The processing of the fusion proteins into mature lipopeptides was determined by jj vivo labelling experiments with tritiated palmitate ( [3H]-palmitate) . This compound was added at a concentration of 50 μCi/ml to early exponential phase cultures ( , = 0.3 - 0.5) of the transformants. Incubation for 10 min was followed by induction of pTac with the addition of 1 mM IPTG and further incubation for 30 min.

Total cell proteins were then precipitated with 10% (w/v) trichloroacetic acid (TCA) for 30 min. at 4° C. The protein pellets obtained by centrifugation at 18,000 g for 15 minutes were washed twice in methanol to remove lipids. The dried pellets were resuspended in sample buffer and analyzed by electrophoresis on discontinuous Tricine-Sodiu Dodecyl Sulfate (SDS) polyacrylamide gels for the separation of low molecular weight proteins following the method described by Schagger and Von Jagow in (1987) Anal. Bioche 166. 368-379. The labelled protein bands were detected by fluorography.

Compared to uninduced cells, transformants containing plasmid pKLY4 induced by IPTG synthesized an additional substance, with an apparent molecular weight of approximately 3000 daltons, constituting the most abundant [Η]-palmιtate-containing compound. Similarly, transf- ormants with the fusion plasmid pKLY3 produced, upon induction, a palmitate-containing compound of less than 2,300 daltons. These two compounds were not found in cells containing the vector pKLY2 independently of induction. The inducible products of pKLY3 and pKLY4 would be translocated across the cytoplasmic membrane as indicated by the loss of mature products in cells treated with globomycin (an inhibitor of signal peptidase II) for 5 min before adding [³H]-palmitate in in vivo labelling experiments. The results showed the presence of a precursor form of the induced product from PKLY3.

The nature of the lipid-modi ied products of pKLY3 and pKLY4 was analyzed by in. vivo labelling with [³H]-tyrosine of transformed cells grown in the above medium without casamino acids. Tyrosine is at C- terminal end of both peptide derivatives expected. Upon induction with IPTG, tyrosine containing compounds of size corresponding to those of the [ H]-palmitate products of pKLY3 and pKLY4 were found. The unique bands found for the lipopeptides labelled with either [Η]-palmitate or [^JH]-tyrosine indicated their chemical stability.

The coding region of the mature alkaline phosphatase gene of E. coli was inserted in frame immediately downstream of the tyrosine codon of the mature colicin E lysis gene on pKLY3. This fusion created plasmid pKLY5. As shown by Manoil and Beckwith in (1986) Science 233: 1403-1408, alkaline phosphatase provides a simple way to monitor the level of expression and secretion of fusion products since it is active in cells only when present in the periplasm. Upon induction with IPTG, DHδαF'IQ transformants containing plasmid pKLY5 produced an enzymatically active pal itate-containing derivative of CQANY-alkaline phosphatase. This product had an apparent molecular weight of approximately 50,000 daltons on a discontinuous Tricine-SDS polyacrylamide gel and was absent from transformants treated with globomycin.

The fractionation of IPTG induced transformants containing PKLY4 or PKLY5 into periplasmic and intracellular, outer membrane, and cytoplasmic membrane components was performed by standard procedures. The radiolabelled cells were harvested and suspended in 10 mM sodium phosphate (pH 7.0). The cells were disrupted by two passages through a French pressure cell at 20,000 lb/in . The cell lysate was centrifuged at 4,500 X g for 10 min at 4°C, and the cell envelope fraction was obtained by centrifugation at 100,000 X g for 40 min at

4°C. The cytoplasmic membrane was solubilized differentially with the detergent sodium lauryl sarcosinate. The outer membrane was sedimented by centrifugation at 100,000 X g for 40 min at 4°C and solubilized in water. Analysis of the TCA precipitated and methanol washed components of the various fractions by electrophoresiε on discontinuous Tricine-SDS polyacrylamide gels showed that the lipid derivatives of CQANY-P2 and of CQANY-alkaline phosphatase were mostly associated with the outer membrane. Thus, the lipid modified polypeptides synthesized in E. coli were secreted across the cytoplasmic membrane. In the periplas , these products would become associated, probably unspecifically, via anchoring of their fatty acids in the lipid layer of the outer membrane. An E. coli rec A lac I— 9 strain DH5αF'IQ transformed with the fusion plasmid pKLY3 was deposited at the American Type

Culture Collection, 12301 Parklawn Drive, Rockville, MD. 20852, U.S.A. under accession number .

A recombinant plasmid similar to pKLY4 in which the coding sequence for the tetrapeptide QANY was replaced by that of IEGR, a specific recognition cleavage site for factor Xa protease, was constructed. The change in the nature of the ø-turn forming tetrapeptide had no effect on the in vivo production of a stable lipid modified derivative of peptide P2. Cleavage of the IEGR-containing lipopeptide by factor Xa protease should allow the release of the free P2 peptide or any other peptide of interest.

Claims

1. A fusion plasmid for in vivo synthesis of a lipid modified polypeptide, said plasmid comprising a first DNA sequence encoding a lipoprotein signal peptide, and at least the first amino acid of a mature lipoprotein, whereby a second DNA sequence encoding said polypeptide can be inserted into said plasmid to produce the desired lipid modified polypeptide.

2. A fusion plasmid according to claim 1, wherein said lipoprotein signal peptide is a bacterial lipoprotein signal peptide.

3. A fusion plasmid according to claim 1, wherein said first DNA sequence encodes a lipoprotein signal peptide, a cysteine and an amino acid sequence with a jδ-turn or an exogenous protease recognition sequence.

4. A fusion plasmid according to claim 1, wherein said lipoprotein signal peptide contains a positively charged amino terminal region, a central hydrophobic region and a carboxyl-terminal region comprising a recognition site for signal peptidase II.

5. A fusion plasmid according to claim 1, wherein said lipoprotein signal peptide is the lipoprotein signal sequence of the ColE₂-plasmid coded lysis protein.

6. A fusion plasmid according to claim 3, wherein the portion of said first DNA sequence encoding the amino acid sequence contributing to the formation of a /3-turn is tetrapeptide QANY.

7. A fusion plasmid according to claim 6, wherein the ,3-turn is conferred at the SPasell cleavage site of said lipoprotein signal peptide.

8. A recombinant fusion protein comprising a polypeptide and at least the first amino acid of a mature lipoprotein, said amino acid having attached thereto at least one fatty acid.

9. A recombinant fusion protein according to claim 8, wherein said first amino acid is a cysteine residue bearing fatty acids at its sulfhydryl and α-NH₂ groups.

10. A recombinant fusion protein according to claim 8, further comprising a short amino acid sequence comprising a 3-turn and the recognition sequence of an exogenous protease.