Dictyostelid expression vector and method for expressing a desired protein

The invention relates to recombinant DNA molecules comprising a Dictyostelium discoideum homologous promoter region, a heterologous DNA sequence with a Dictyostelium discoideum homologous peptide sequence capable of functioning as a leader peptide sequence positioned upstream thereof and in proper reading frame therewith, and a Dictyostelium discoideum homologous termination region, the heterologous DNA sequence encoding the desired functional polypeptide or intermediate thereof, said DNA sequence being positioned downstream from the promoter region and said termination region being positioned downstream from the DNA sequence. The invention also provides recombinant dictyostelid hosts as well as methods for preparing recombinant DNA molecules of the invention and for expressing recombinant polypeptides encoded by said recombinant DNA molecules in dictyostelid hosts. The invention in particular relates to the expression of the CS protein of Plasmodium falciparum.

FIELD OF THE INVENTION 
The present invention relates to recombinant DNA molecules and a method for 
expressing desired DNA sequences in dictyostelids. More in particular it 
relates to a recombinant plasmid and method for expressing a desired 
enzyme or parasite antigen in dictyostelids and more in particular to 
expressing the circumsporozoite antigen of Plasmodium falciparum and 
chloramphenicol acetyl transferase in Dictyostelium discoideum. 
BACKGROUND OF THE INVENTION 
Production of functional or immunogenic recombinant polypeptides has been 
obtained in multiple organisms. In general, the gene of interest has been 
isolated and expressed under the control of specific DNA elements allowing 
expression in the particular host. Bacteria, lower and higher eukaryotic 
cells and viral vectors have been among the most widely used systems. 
Plasmodium falciparum, the most frequent human malaria cadsatire parasite, 
is found in different forms in insect and human hosts. The use of 
inactivated parasite forms as vaccine in mammals have shown promising 
results. A limitation, however, being the low amount of material available 
due to the difficulty of cultivating parasites. The genes coding for some 
cell surface proteins of the different forms have been sequenced allowing 
identification of host protective antigens possibly useful as vaccines. 
Expression of such antigens or other P. falciparum proteins in 
heterologous recombinant systems (e.g. E. coli, yeast, vaccinia virus, 
baculovirus, salmonella) has been difficult, and in most instances, only 
small amounts of complete proteins were obtained. 
The surface of the first form of Plasmodium falciparum found in the 
organism after insect bite transmission, is covered by the 
circumsporozoite (CS) protein. This protein is synthesized in the form of 
a polypeptide precursor which is composed of an amino terminal signal 
sequence removed upon processing, of a large central repeat domain flanked 
on both sides by regions referred as region I and II containing conserved 
sequences between different Plasmodia species and of a terminal carboxy 
anchor domain. The repeat domain consisting of (ASN-ALA-ASN-PRO).sub.n has 
been shown to be the B-cell immunodominant region of the P. fatciparum CS. 
Synthetic peptides containing such a repeat have been used with limited 
success as subunit vaccine in protection studies. The T-cell response 
elements on the CS protein have been mapped outside of the repeat 
segments. These results suggest that the entire CS protein could be used 
to obtain a stronger and longer lasting immune protection. These 
experimental data further indicate the requirement for a protein with its 
B- and T-cell epitopes to obtain an efficient malaria vaccine. 
Difficulties in producing high amounts of CS protein were bypassed by 
expressing only segments of the CS protein in vivo which, however, did not 
elicit a sufficient immune response as vaccines. Expression of the 
complete protein was obtained using vaccinia and baculovirus expression 
systems. However, unstable expression of proteins as well as the high cost 
of production render such systems unattractive. 
It is therefore an object of the present invention to provide an expression 
system for different kinds of proteins, such as Plasmodium falciparum 
proteins, that is capable of stable expression of proteins at low cost. 
DETAILED DESCRIPTION OF THE INVENTION 
It has been found that species of the slime mold Dictyostelium can be used 
as an efficient eukaryotic expression system for the production of 
recombinant proteins. 
The cellular slime mold Dictyostelium discoideum (Dd) is a free-living 
organism, easy to grow and to maintain. Dd strains can grow on bacteria 
lawns with a doubling time of about 3 hours, in bacterial suspensions to 
high densities (up to 10.sup.10 cells per liter) or in semi-synthetic 
media containing glucose, peptone and yeast extract where doubling time is 
about 12 hours. The life cycle of Dictyostelium consists of a growth and a 
developmental phase. The developmental phase is triggered by starvation 
and is characterized by aggregation of previously single cells to form a 
multicellular organism which then differentiates to produce spores which 
can be stored over a prolonged period of time. Germination of spores in 
the presence of bacteria or rich medium will allow renewed growth. During 
this developmental cycle, diffusible factors are produced and for at least 
one of them (cAMP) binding to its receptor induces transcription of a set 
of specific genes (See Loomis, The development of Dictyostelium 
discoideum, Acad. Press, 1982). 
Growth properties and transformation capacity of Dictyostelium discoideum 
offers the possibility to express foreign proteins, since cells can be 
grown at low cost on bacteria and expression of specific proteins can be 
tightly controlled by starvation in a simple medium. 
In one embodiment of this invention the homologous promoter region is the 
Discoidin I promoter of Dictyostelium discoideum which is under 
developmental control. Transcription from this promoter is induced by 
starvation of the Dictyostelium cell culture. 
Furthermore a leader peptide sequence SEQ ID NO: 1! is provided in the 
expression vector. The DNA sequence of SEQ ID NO: 1 inherently encodes for 
the following amino acid sequence (SEQ ID NO: 3): 
Met Ser Arg Phe Leu Val Leu Ile Ile Leu Tyr Ash Ile 
Leu Asn Ser Ala His Ser Ala Pro Thr Gln Asp Pro 
Fusion of this leader peptide allows export of the recombinant protein to 
the cell surface thus facilitating recombinant protein identification. 
In another embodiment the invention provides an inducible expression 
vector. The Dd ras promoter SEE SEQ ID NO: 2! of Dictyostelium discoideum 
is used there as the homologous promoter region. Expression of genes under 
the control of the Dd ras promoter is triggered by cAMP addition to the 
cell culture. During cell growth the transcription level from the Dd ras 
promoter is undetectable, thus allowing to introduce genes encoding 
potentially toxic proteins in Dictyostelium. The production of these 
proteins is triggered by cAMP addition during development.

The invention is further illustrated by the following examples, which 
should not be considered as limiting the scope of the present invention. 
A deposit of Dictyostelium discoideum designated by accession No. CBS 
238.91 was made with Centraalbureau voor Schimmelcultures, Oosterstraat 1, 
P.O. Box 273, 3740 AG BAARN, The Netherlands, on Aug. 22, 1991, 
depositor's reference designation pEDII-CS. 
EXAMPLES 
The following examples utilize many techniques well known and accessible to 
those skilled in the art of molecular biology. Such methods are not always 
described in detail. 
Enzymes are obtained from commercial sources and used according to the 
supplier's protocols. 
Bacterial media and current cloning techniques are described in Sambrook et 
al. (Molecular cloning: A laboratory manual, CSH Press, 1989.) 
Monoclonal antibodies and NANP.sub.50 peptide were obtained from H. Matile 
(Hoffman La Roche Ltd.). 
Example 1 
The following examples teach the expression of the circumsporozoite antigen 
CS of Plasmodium falciparum in Dictyostelium discoideum under the control 
of the Discoidin I promoter. 
1.1. Construction of CS containing plasmids. 
Expression vector pEDI-CS is constituted of the pVEII vector (Maniak and 
Nellen, Nucl. Acids Res., 18, 5375, 1990), which contains the elements 
important for propagation and maintenance in a prokaryotic host (origin of 
replication and ampicillin resistance), and of a Tn903 encoded neomycin 
resistance gene conferring geneticin (G418) resistance to eukaryotic cells 
under the control of a Dictyostelium actin 15 transcription unit. The 
presence of a Discoidin I promoter allows the developmental control of 
expression of downstream sequences and actin 8 sequences insure proper 
termination of the RNA. 
For construction of the pEDI-CS expression vector the HaeIII+RsaI 
restriction fragment of 1161 bp of the CS NF54 gene (Caspers et al., Mol. 
and Biochem. Parasitol 35, 185, 1989) was first inserted onto the 
Asp718+BamHI site of pVEII filled in by Klenow DNA polymerase. 
Subsequently both DNA strands of a sequence encoding the contact site A 
(CsA) leader peptide plus 3 amino acids were synthesized on a Applied 
Biosystem Model 380 B DNA synthesizer. The nucleotide sequence of the 
synthetic leader peptide SEQ ID NO: 1! (FIG. 1) was confirmed by 
introducing the blunt end fragment onto M13mp18 replicative form at the 
SmaI site, followed by DNA sequencing. 
The XbaI+BamHI restriction fragment containing CsA leader peptide was then 
isolated and inserted at the XbaI+Bam HI sites present in the vector to 
generate expression vector pEDII-CS. 
1.2. Transformation of Dictyostelium discoideum. 
Dictyostelium cells were cultured in shaking suspensions in HL-5 medium up 
to a concentration of 2-5.times.10.sup.6 /ml, centrifuged at 300 g and 
rinsed in distilled water. 5 .mu.g of the desired DNA was electroporated 
onto 10.sup.7 cells in 100 .mu.l of distilled water at 670 V and 3 .mu.F 
using a Gene Pulser apparatus from BioRad, thus delivering a pulse of 
about 1.5 msec. 
Progressive G418 selection was applied to the cells up to a concentration 
of 50 .mu.g/ml, ensuring the presence of 100 to 200 copies of tandem 
repeats of inserted DNA. 
1.3. CS expression upon starvation at the RNA level. 
RNA was prepared from cells grown in HL-5 medium (vegetative stage) or 
after 4, 8 or 15 hours of starvation in PDF, respectively. 
Therefor 10.sup.7 cells were lysed in 50 mM Tris pH 8.5, 0.1% SDS, 1 mM 
vanadyl complex and extracted 3 times with phenol-chloroform 1:1. 
15 .mu.g of RNA were treated with glyoxal and subjected to electrophoresis 
on a 0.8% agarose gel. The samples were transferred to Genescreen plus 
filters with a vacugene apparatus. Anti-sense RNA probes were generated by 
inserting DNA fragments onto the vector pGEM-1, and carrying out Sp6-RNA 
polymerase reactions. The filters were hybridized for 48 hours at 
55.degree. C. in a solution containing 50% formamide, 5XSSC (1XSSC=0.15M 
NaC1, 15mM sodium citrate), 0.2% bovine serum albumin, 0.2% Ficoll, 0.2% 
polyvinylpyrrolidone, 25mM Na.sub.2 - HPO.sub.4, 25mM NaH.sub.2 PO.sub.4, 
0.2% SDS, 1mM EDTA, 250 .mu.g/ml denatured DNA and 500 .mu.g/ml yeast RNA. 
The filters were washed 5=15 min. in 0.1XSSC, 0.12% SDS at 65.degree. C. 
On the Northern blots, an RNA species of 1.4 kb was detected in pEDII-CS 
transfected cell, whereas no signal was detected in pVEII transfected 
cells. The amount of steady-state CS RNA increases after 4 hours of 
starvation, followed by a decrease in expression after 15 hours. 
1.4. CS expression upon starvation at the protein level. 
Proteins were prepared from the same aliquots used in example 1.3. for the 
analysis of CS expression upon starvation at the protein level. 
Cells were lysed in 1 X Laemmli's buffer at 100.degree. C. for 5 min. and 
proteins were separated on a 10% SDS-PAGE. Proteins were 
electrotransferred onto a nitrocellulose filter. 0.2 mg/ml of the 
anti-NANP monoclonal antibody was added to the filter for an overnight 
incubation at room temperature. .sup.125 I-protein A was used to reveal 
the anti-NANP reaction. 
The Western blots showed no CS expression in pVEII cells and a maximal 
expression in pEDII-CS cells after 4 hours of starvation. A single band of 
62 kDA molecular mass was detected. No other crossreacting species was 
observed. This induction of the expression follows the profile expected 
for Discoidin I gene regulation. 
1.5. Recognition of the NANP epitope on the CS protein. 
To confirm recognition of the NANP epitope present on the CS protein, the 
binding of an anti-NANP monoclonal antibody to the Dd expressed CS protein 
was competed with a NANP.sub.50 peptide. Cells expressing the CS 
polypeptide were lysed, proteins separated by SDS-PAGE and transferred 
onto a nitrocellulose filter. Before incubation, the anti-NANP.sub.50 
monoclonal antibody was incubated with different amount of a NANP.sub.50 
peptide which correspond to the number of NANP.sub.50 repeats present on 
the CS protein. A tenth equimolar amount of NANP.sub.50 peptide was 
sufficient to observe a significant reduction in the binding of the 
monoclonal antibody to the CS protein. 
1.6. Recognition of different Plasmodium falciparum CS epitopes by 
monoclonal antibodies. 
Different monoclonal antibodies against CS proteins or synthetic peptides 
were used in the immunoblot assay. Dd cells were lysed, proteins separated 
by SDS-PAGE and transferred onto a nitrocellulose filter. Filter strips 
were incubated with different monoclonal antibodies from various 
categories (SP3B4, SP3.E9, CT3.3, CT3.1, SP3.H3, SP3.E6, SP3.C6). 
Recognition of epitopes was visible only with the expected antibodies. In 
no cases, a specific signal was observed in cells expressing pVEII vector 
only. 
1.7. Purification of CS protein produced in Dictyostelium discoideum. 
At the amino acids level, the CS protein contains a carboxy terminal 
hydrophobic segment which could play a role as a signal for the addition 
of a glycosyl-phosphatidy-linositol anchor (GPI). This C-terminal peptide 
segment or a posttranslationally added GPI anchor should confer a 
hydrophobic nature to the CS protein thus allowing its partitioning in 
Triton X-114. 
Proteins were extracted from the cells with 1% of Triton X-114 and 
separated into aqueous and detergent phases. The detergent soluble 
proteins were analyzed by Western blot. At least 10 times more proteins 
were present in the aqueous phase than in the TX-114 phase estimated by a 
Biorad Protein assay and also as judged by Ponceau staining. A strong 
signal is observed in the TX-114 phase of CS expression cells, even though 
a certain amount of protein still remains in the aqueous phase. No signal 
is found in the pVEII sample. Treatment with Triton X-114 therefore allows 
a first partial purification of the recombinant protein. 
In order to further purify CS protein expressed in Dictyostelium, CS cells 
were pulse-labelled with .sup.35 S!-methionine for 2 hours after an 
initial starvation period of two hours. Cells were removed by 
centrifugation, lysed in Crumpton lysis buffer and precleaned with protein 
A Sepharose. TX-114 soluble proteins were loaded on an affinity column 
containing anti-NANP antibody crosslinked to Sepharose. After elution from 
the column in the presence of 0.1 M glycine (pH 2.5) methionine-labelled 
protein with an apparent molecular weight of 62kDa was detected by 
SDS-PAGE and fluorography. The same experiment performed on pVEII 
containing cells revealed only minor bands of different molecular weights 
(FIG. 3). 
1.8. Cell surface expression. 
If correctly processed, the CS protein should be present on the cell 
surface. Analysis of fluorescent activated cell sorting using an anti-NANP 
antibody showed that the CS protein is expressed at the surface of 
Dictyostelium discoideum cells. 
1.9. Terminal sequencing. 
In order to determine whether the expressed CS protein was complete, CS 
protein was isolated, using Triton TX-114 phase separation and 
immunoaffinity chromatography, to sequence its N-terminus. 
No sequence could be obtained from the complete protein, indicating a 
probable block at the amino terminus. When the purification was done in 
presence of fewer protease inhibitors, an amino acid sequence missing the 
first 25 amino acids was obtained. When only benzamidine was used up to 33 
amino acids were absent from the N-terminus suggesting that endogenous 
protease could attack the CS protein during its purification. As shown, 
this problem can be solved by using protease inhibitors and is limited to 
the first 20 amino acids. 
Example 2 
The following example teaches the expression of the circumsporozoite 
antigen CS of Plasmodium falciparum in Dictyostelium discoideum fused to a 
leader peptide from contact site A and under the control of an inducible 
promoter (Dd ras) 
2.1. DNA-sequence of the Dd ras promoter 
The DNA-sequence of the Dd ras promoter used in this example is identical 
to nucleotides 1 to 401 SEE SEQ ID NO: 2! from FIG. 4 (see also example 
3) 
2.2. Construction of pERI-CS vector 
The Discoidin I promoter region of pEDII-CS was excised using PacI 
resriction digestion, leaving 68 nucleotides of transcribed but 
untranslated Discoidin I sequence, the AUG codon, the contact site A 
leader peptide and the CS coding region (see FIG. 2). A Dd ras promoter 
fragment encompassing nucleotides 1 to 401 of FIG. 4 was obtained by 
exonuclease III digestion and addition of BamHI linkers. After blunt 
ending with T4-polymerase, this fragment was ligated in the above 
mentioned PacI digested CS vector to obtain pERI-CS 
2.3. Expression of the CS protein 
The construct pERI-CS of example 2.2. was reintroduced into Dictyostelium 
and cells analyzed for CS expression by Western blot (see example 1.4.; 
results not shown). CS expression was very low in vegetative cells starved 
in suspension in PDF. An increase of about 100 fold was observed in the 
level of CS protein already after one hour of cAMP addition (0.2 mM). Two 
hours of induction further increased expression whereas the level of CS 
protein decreased after 16 hours of incubation. 
Example 3 
The following example teaches the expression of chloramphenicol acetyl 
transferase in Dictyostelium discoideum using an inducible expression 
vector. 
3.1. DNA sequence of the Dd ras promoter. 
The RsaI restriction fragment from the genomic clone of Dd ras was inserted 
into the SmaI site from pGEM1. Sequencing was performed using specific 
primers on double stranded DNA. 
FIG. 4. shows the DNA sequence of the RsaI fragment. Arrows indicate 
transcription start sites. The thick arrow corresponds to the major 
transcript detected upon external cAMP addition. Dotted arrows indicate 
further RNA start site used during normal Dd development. Met is the Dd 
ras protein start codon. The region essential for promoter function is 
underlined. * corresponds to the breakpoint of a 3' deletion providing a 
DNA fragment comprising nucleotides 1 to 434; ** indicates the breakpoint 
of a 3' deletion providing a DNA fragment comprising nucleotides 1 to 401; 
*** corresponds to the breakpoint of a 5' deletion providing a DNA 
fragment comprising nucleotides 418-630. 
3.2. Construction of the CAT containing vector. 
Parts of Dd ras promoter sequences were placed in pAV-CAT vector (May et 
al., Mol. Cell Biol. 9, 4653, 1989). The resulting constructs resemble 
pEDII-CS with CS and leader peptides replaced by a chloramphenicol acetyl 
transferase gene (CAT), Discoidin I promoter by Dd ras promoter and the 
G418 resistance gene under the control of the actin 15 promoter and 
terminator region placed in opposite orientation. 
3.3. Expression of chloramphenicol acetyl transferase in Dictyostelium 
discoideum on the RNA level. 
The constructs of example 2.2. were reintroduced in Dictyostelium and cells 
analyzed for CAT expression on the RNA level. 
RNA was prepared from Dd cells transformed with a recombinant pAV-CAT 
vector containing parts of the RsaI fragment of the Dd ras promoter. RNA 
was isolated from cells starved for 7 hours. Half of the cells received 
cAMP (0.2mM) for 1 hour before RNA preparation. After separation on 
agarose gel (MES-glyoxal) and transfer to Genescreen, the RNA was probed 
with pAV-CAT whole vector labelled by nick translation. 
CAT transcripts were detectable after cAMP induction with Dd ras promoter 
fragments containing at least the sequence underlined in FIG. 4. Further 
deletions abolished CAT RNA accumulation. Optimal CAT transcript 
accumulation occurred with a DNA containing sequence 1 to 401 (** in FIG. 
4). 
3.4. Expression of chloramphenicol acetyl transferase in Dictyostelium 
discoideum at the enzyme level. 
10.sup.7 Dd cells containing pAV-CAT vectors with nucleotides 1 to 434, 1 
to 401 and 418 to 630 SEE SEQ ID NO: 2! respectively were lysed by 3 
cycles of freezing and thawing. After centrifugation at 12'000 rpm for 5 
min. CAT activity in supernatant was assayed for 30 min. by described 
techniques (Gorman et al., Mol. Cell. Biol. 2, 1044, 1982). Due to large 
amounts of CAT activity in the extracts, only 1 .mu.l out of 50 .mu.l of 
supernatant was used to insure being in the linear phase of the reaction. 
CAT activity assays showed not only the presence of a functional enzyme in 
Dd extracts, but also induction with the proper constructs. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 77 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: DOUBLE 
(D) TOPOLOGY: UNKNOWN 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
ATGTCTAGATTTTTAGTATTGATAATATTATATAATATT39 
TTAAATAGTGCACATTCAGCTCCAACCCAGGATCCATG77 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 731 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: UNKNOWN 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
GTACATAATATTTTTGTGTTCTTATAATTTGGTTAAATCGATGAATAATA50 
TTTGATTAGTATATGTTTTTTTTTCCTTTTTTTTATTTTTATTTTTATTT100 
TTTTAAAAAATAAAAATTAGAATAAAATATTTCTATTTGAAGGAGTTTTT150 
ATTTGTATTTAAAATTATATTAAACATAGTGAACCTAAAAATAGATTTGT200 
GACGGTATATGATAAGAAAATTCTAAAAAAAAAATTCAGATAATTTTTGG250 
ATTGGAAACAACAACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCAAA300 
AAAAAAAAAAAAAAAATTAAAATCAAAAAAAAAAGGTATTTAAAGAAATT350 
TTTTAAAATATTATTATATATCTTTAATTGTGCAAAACACACTTTTAACA400 
CACTCTATTATCTTACAAAGGTTTAAAATTTTAATTTTTTTTATTTAATT450 
ATTATTTTTTTAAATAAATTTTTTTTAATTTTTTAATTTTTTTTTTTTTT500 
TTACCATCAACCCCTTTAATCAAACAAATAACATTTATTATTTATTTATT550 
TTATATATATCAATTAGAAATAAAAATATTTTCCTAATAGTAGTAATAAT600 
AATTTCTTTTTAATAAAAATACCTTTTTCTACATTATTATTTTTTTATTA650 
TTTTTTTCTTTAATCATTCAAAATTTTATTTTTTTTTTTAAAAAAAAAAA700 
AACAATTAAAACAAACAATTTAAAAAAAATG731 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 
(B) TYPE: AMINO ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: UNKNOWN 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
MetSerArgPheLeuValLeuIleIleLeuTyrAsnIleLeuAsn 
151015 
SerAlaHisSerAlaProThrGlnAspPro 
2025 
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