WO1993006125A1

WO1993006125A1 - ISOLATION AND CHARACTERIZATION OF cDNA OF PLASMODIUM FALCIPARUM GLUCOSE-6-PHOSPHATE DEHYDROGENASE

Info

Publication number: WO1993006125A1
Application number: PCT/US1992/007807
Authority: WO
Inventors: David C. Kaslow; Mohammed Shahabuddin
Original assignee: The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority date: 1991-09-20
Filing date: 1992-09-21
Publication date: 1993-04-01
Also published as: AU2592692A

Abstract

DNA segments encoding the Plasmodium falciparum glucose-6-phosphate dehydrogenase protein obtained by polymerase chain reaction techniques.

Description

ISOLATION AND CHARACTERIZATION

OF cDNA OF PLASMODIUM FALCIPARUM

GLUCOSE-6-PHOSPHATE DEHYDROGENASE

1. Field of the Invention The present invention relates to glucose-

6-phosphate dehydrogenase from Plasmodium falciparum and to the DNA segment which encodes it.

2. Background Information

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway. In most organisms the pathway has two main functions: production of pentose (ribose) for biosynthesis of nucleic acids and several coenzymes, and reduction of NADP for a variety of detoxification and reductive biosynthetic reactions. Recently, Vander Jagt et al. reported that isocitrate dehydrogenase may be responsible for providing much of the NADPH required for reductive biosynthesis within the Plasmodium falciparum parasite (D.L. Vander Jagt, L.A. Hunsaker, M. Kibirige, N.M. Campos, Blood. 74, 1, 471-474 (1989)); while, Roth et al. reported that the majority of ribose synthesis in parasite infected red blood cells (RBCs) appears to occur through pathways other than those involving G6PD (E.F. Roth, R.M. Ruprecht, S. Schulman, J. Vanderberg, J.A. Olson, J. Clin. Invest.. 77, 1129-1135 (1986)). Therefore, consistent with the findings of Usanga and Luzzatto, parasite encoded G6PD does not seem necessary for parasite survival in normal erythrocytes (RBCs) (E.A. Usanga, L. Luzzatto, Nature. 313, 793-795 (1985)).

Several investigators have reported that when cultured in G6PD deficient RBCs, P. falciparum

HEET parasites initially have a reduced growth rate, but following an adaptation period, the growth again approximates in vivo rates (Usanga et al. (1985) ; I.T. Ling, R.J.M. Wilson, Mol. & Biochem. Parasit. , 31, 47-56 (1988)); E.F. Roth, C. Raventos-Suarez, A. Rinaldi, R.L. Nagel, PNAS. 80, 298-299 (1983)); and E.F. Roth, S. Schulman, Brit. J. Hema.. 70, 363-367 (1988) . Production of parasite G6PD following a lag phase seems to fully explain the recovery of normal growth rate during persistent culture in G6PD deficient erythrocytes (Usanga et al. (1985)). However, it has been subsequently observed (Ling et al. (1988); Roth et al. (1983); Roth et al. (1988); and B. Kurdi-Haidar, L. Luzzatto, Mol. & Biochem. Parasit.. 41, 83-92 (1990)) that the parasite expresses G6PD constitutively, even in G6PD normal RBCs. The mechanism by which the parasite recovers to normal growth within a few cell cycles in G6PD deficient RBCs, and the mechanism that confers relative protection against malaria in females heterozygous for G6PD deficiency, despite expression of parasite encoded G6PD, now remain an even more perplexing enigma.

Further characterization and subcellular localization of the parasite encoded G6PD may provide clues as to how the parasite adapts in homozygous or hemizygous G6PD deficient erythrocytes, yet apparently fails to adapt in female mosaic. Such further characterization and localization may also lead to a new class of chemotherapeutic agents effective against the ever increasing population of drug resistant malaria parasites. To this end the P. falciparum glucose- 6-phosphate dehydrogenase gene has been isolated and sequenced (and expressed in Escherichia coli) . Given the strong genetic and epidemiological evidence linking human G6PD deficiency with protection from malaria, and widespread resistance to current chemotherapeutic agents, development of a new class of agents directed against the potential "achilles heel" of the parasite was the impetus for the research that lead to the cloning of G6PD.

SUMMARY OF THE INVENTION It is an object of the present invention to characterize the molecular structure of the glucose-6-phosphate dehydrogenase enzyme of Plasmodium falciparum in order to better design and exploit chemotherapeutic agents against malaria. Accordingly, the present invention relates to DNA segments encoding glucose-6-phosphate dehydrogenase in Plasmodium falciparum .

The present invention additionally relates to the amino acid sequence of Plasmodium falciparum glucose-6-phosphate dehydrogenase.

Various other objects and advantages of the present invention will become obvious from the figure and the following description of the invention. All publications mentioned herein are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the nucleotide sequence (SEQ ID N0:1) of the cDNA encoding Plasmodium falciparum glucose-6-phosphate dehydrogenase protein.

SUBSTITUTE SHEET Figure 2 shows the deduced amino acid sequence (SEQ ID NO:2) of the protein encoded by the cDNA of Figure 1.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a cDNA clone isolated by polymerase chain reaction techniques which encodes the glucose-6-phosphate dehydrogenase protein from Plasmodium falciparum. The isolated cDNA clone can be obtained in a substantially pure form by using conventional methods used by those of ordinary skill in the art. The present invention also relates to the glucose-6-phosphate dehydrogenase protein from Plasmodium falciparum encoded by the cDNA. The protein has a novel structure as compared to all other (human, rat, fruit fly, yeast, and E. coli) G6PD deduced amino acid sequences. Although the predicted NADP binding site and glucose-6-phosphate binding site is conserved, the P. falciparum enzyme apparently has a secretory signal sequence, a membrane spanning segment, and a transmembrane helix, none of which are found in other G6PD deduced amino acid sequences.

The present invention further relates to a recombinantly produced P. falciparum G6PD protein with the amino acid sequence given in Figure 1, plus any allelic and/or biologically functioning variants of this sequence, or any unique portion of this sequence. The recombinant protein can be expressed in a number of expression systems, including both bacterial and eukaryotic. Further, the present invention relates to a synthetic P. falciparum G6PD protein. The present invention relates to a recombinant DNA molecule comprising a vector and a DNA segment encoding the P. falciparum G6PD protein. Using methodology well known in the art, recombinant DNA molecules of the present invention can be constructed. Possible vectors for use in the present invention include, but are not limited to pUC 13, pUC 19, pcDNAII, pBluescriptll. The DNA segment can be present in the vector operably linked to regulatory elements, including, for example, a promoter.

The invention further relates to host cells comprising the above-described recombinant DNA molecule. The recombinant DNA molecule may be stably transformed, stably transfected, transiently transfected into the host cell or in alive attenuated virus. In each case, the host cell expresses a functionally active form of the protein encoded by the recombinant DNA molecule. The host cells used can be either bacterial or eukaryotic. Some non-limiting examples of bacterial host cells are Escherichia coli and Staphylococcus aureus . Non-limiting examples of eukaryotic host cells are Saccharomyces cereviεiae , CHO cells, COS cells, and Sf9 cells. Transformation with the recombinant molecules can be effected using methods well known in the art.

The present invention further relates to a method of screening drugs for anti-malarial activity by contacting a drug to the recombinant P. falciparum G6PD protein under conditions such that inhibition of said P. falciparum G6PD activity can be effected. (See D.C. Kaslow and S. Hill, JBC. 265, 21, 12337-12341, 1990.) By means of such drug screeing assays, the striking structural

SUBSTITUTE SHEET features of the amino acid sequence of the protein can be exploited in the design of a chemotherapeutic intervention for malaria. The strong genetic and epidemiological evidence that human G6PD deficiency affords protection against malaria further suggests that malaria parasite G6PD may be a rational target for drug therapy.

Comparative assays were conducted to determine G6PD activity in the transfected cells which had been contacted with a drug versus G6PD activity in uncontacted transfected cells. After being contacted with the drug, the cells were placed in an environment where labeled glucose was the only source of carbon. Comparative assays were also conducted with untransfected cells as a control.

The effect of the drug on the transfected cells was detected by measuring the presence of labelled PfG6PD reaction product. (Please correct and/or add further details to this Paper Example.) The present invention further relates to antibodies specific for the P. falciparum G6PD protein of the present invention. One skilled in the art, using standard methodology, can raise antibodies (such as monoclonal, polyclonal, anti- idotypic and monoclonal catalytic [Sastry et al.

PNAS 86:5728-5732 (1989)]) to the P. Jralciparum G6PD protein, or a unique portion thereof. In a further embodiment, such antibodies can be used in assays to detect the presence of P. falciparum G6PD protein in serum from a patient suspected of being infected with P. falciparum. Antibodies specific for the P. falciparum G6PD protein or a unique portion thereof can be coated on to a solid surface such as a plastic and contacted with the serum sample. After- washing, the presence or absence of the protein from the serum bound to the fixed antibodies is deteted by addition of a labeled (e.g. fluorescently labeled) antibody specific for the P. falciparum G6PD protein. One skilled in the art will appreciate that the invention includes the use of competition type assays in detecting in a sample the antigens to which this invention relates.

The present invention also relates to a vaccine for use in humans against malaria. As is customary for vaccines, the P. falciparum G6PD protein, or a unique portion thereof, can be delivered to a human in a pharmacologically acceptable vehicle. As one skilled in the art will understand, it is not necessary to use the entire protein (for example, a synthetic polypeptide corresponding to the P. falciparum G6PD protein) can be used. Pharmacologically acceptable carriers commonly used in vaccines can be used to deliver the protein or peptide. Such carriers include MTP, tetanus toxoid or liposomes. Vaccines of the present invention can include effective amounts of immunological adjuvants known to enhance an immune response. Such adjuvants include IL-2 and alum. The protein or polypeptide is present in the vaccine in an amount sufficient to induce an immune response against the antigenic protein and thus to protect against Plasmodium infection thereby protecting the human against malaria. Protective antibodies are usually best elicited by a series of 2-3 doses given about 1 to 6 months apart. The series can be repeated when concentrations of circulating antibodies in the human drops. Further, the vaccine can be used to immunize a human against other forms of malaria (that is, heterologous immunization) .

EXAMPLES For purposes of illustrating a preferred embodiment of the present invention the following non-limiting examples will be discussed in detail.

Parasites and cDNA Library Construction.

The 3D7 clone of P. falciparum isolate NF54 (D. Walliker, I.A. Quakyi, T.E. Wellems, McCutchan, A. Szarfman, W.T. London, L.M. Corcoran, T.R. Burkot, R. Carter Science 236, 1661-1666 (1987)) and the HB3 isolate (Walliker et al. (1987)) were cultured in vitro. Total cellular RNA, purified from stage III to IV 3D7 gametocytes and from HB3 asexual parasites, was used to construct oligo dT primed, size-selected, BstXI linkered cDNA libraries in plasmid pcDNA II (Invitrogen) . The libraries were screened (J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual. 2d Ed. (1989)) with radiolabelled, random primed DNA probes (A.P. Feinberg, B. Vogelstein, Anal. Biochem. 137. 266-267 (1984)).

Polymerase Chain Reaction Degenerate synthetic oligonucleotides were used to amplify the G6PD gene from P. falciparum cDNA or genomic DNA as follows: a sense strand oligonucleotide,

5'-ggaattcAT{ACT}GA{CT}CA{CT}TA{CT} {CT}T{ACGT}GG{ACGT>AA{AG>GA-3 ^» , located 5' of an antisense strand oligonucleotide, 5^•-cggatccTG{AG}TT{TC>TGCAT-CACGT} AC{AG}TC{ACGT}C-3 ' , were paired as primers in a polymerase chain reaction (R.K. Saiki, D.H. Gelfand, S. Stoffel, S.J. Scharf, R. Higuchi, G.T. Horn, K.B. Mullis, H.A. Erlich, Science. 239, 487-491 (1988)). 4 cycles of denaturation at 94°C for 2 minutes, annealing at 37°C for 2 minutes, and extension at 72°C for 1 minute were followed by 25 cycles of denaturation at 94°C for 30 seconds, annealing at 45°C for 30 seconds, and extension at 72°C for 1 minute; amplified DNA was purified and cloned as previously described (Kaslow et al. (1990)).

Northern and Southern Blots

Pulsed field gel electrophoresis was performed as described by Welle s et al. (T.E. Wellems, D. Walliker, C.L. Smith, V.E. Do Rosario,

W.L. Maloy, R.J. Howard, R. Carter, T.F. McCutchan

Cell 49, 633-642 (1987). Southern and Northern blot analyses was performed as described by Kaslow et al.

(D.C. Kaslow, B.R. Migeon, M.G. Persico, M. Zollo, J.L. Vander Berg, P.B. Samollow, Genomics 1, 19-28

(1987) ) .

Cloning the PfG6PD Gene

Attempts to clone the P . falciparum G6PD gene by hybridization with human G6PD cDNA at low stringency or with "guessmers" comprising highly conserved regions, or by complementation in pgi/zwf deficient E . coli (DF214) either on glucose minimal media or on diamide containing rich media have been unsuccessful. Recently, the Saccharomyces cereviεiae G6PD gene was cloned: Thomas et al. cloned the gene by complementation for a defect in inorganic sulfur metabolism (methionine auxotrophy) (D. Thomas, H. Cherest, Y. Surdin-Kerjan, EMBO 10, 547-553 (1991)). S . cerevisiae G6PD gene was also cloned by using the polymerase chain reaction (PCR) with highly degenerate oligonucleotides (I. Nogae, M. Johnston, Gene. 96, 161-169 (1990). When 6 sense and 11 antisense primers were used in PCR, only a single pair of primers was found to yield a fragment of the yeast gene. When this latter pair of primers was used in PCR with genomic yeast DNA or genomic P. falciparum DNA, a product was observed only in the reaction containing yeast DNA template. A further 13 permutations with 9 primers were examined by PCR using P. falciparum DNA as the template. One pair of primers (FIG. 1) amplified a I93bp fragment from P. falciparum DNA. The nucleotide sequence of this fragment differed from the published DNA sequences of human, E. coli , and S . cereviεiae G6PD, but typical of P. falciparum nucleotide sequence, was 74% A+T. In contrast, the deduced amino acid sequence from the fragment showed striking homology to mammalian, yeast, fruit fly, and bacterial G6PD amino acid sequence (FIG. 1) .

P. falciparum gametocytes express parasite encoded G6PD at a high level. Therefore, to clone G6PD cDNA, a gametocyte specific cDNA library constructed in pcDNAII (Invitrogen) was screened with the 193bp PCR product. pPfg6pd2 (wpMS2) was selected for further characterization, and was found to have a 1750 bp insert, but did not contain the full length coding sequence (FIG. 1) . An asexual stage cDNA library was also screened from which several additional clones were isolated. pPfg6pd6 (MS6) contained the most 5¹ sequence.

The insert from pPfg6pd2 hybridized to chromosome 14 by Southern blot analysis of size-fractionated P. falciparum chromosomes, confirming that the cDNA originated from P. falciparum and not human RNA or other potential contaminants.

Sequence Analysis of pfG6PD Universal sequencing primers and synthetic oligonucleotides are used to obtain DNA sequence from double stranded plasmid with Sequenase (United States Biochemicals Corp.). 100% of the sequence was determined from both strands. A 2259 bp open reading frame, encoding an

88 kDa polypeptide of 751 amino acids, was deduced from the nucleotide sequence (FIG. 1) . The presumptive initiation codon is in accordance with the P . falciparum consensus sequence, and the A+τ content of 77% in the predicted coding region, and 85% in the 3' noncoding regions are typical of P. falciparum genes.

Comparison of the cDNA nucleotide sequence with that obtained from cloned genomic restriction enzyme fragments (nucleotide 562-1396) , and comparison of PCR products from genomic DNA to that from cDNA suggest that the gene does not contain introns within this region but rather an insertion of 61 amino acids (residues 268-254) in between residues 111-137 of human G6PD (B. Persson, H.

Jδrnvall, I. Wood, J. Jeffery, FEBS. 1991, 486-491 (1991) . Comparison of the deduced amino acid sequence with previously published human G6PD sequences revealed an overall identity of 39%. The gene encoding P. falciparum G6PD is the first to be isolated in the pentose phosphate pathway from Plasmodia . As the genes encoding G6PD from mammals, insect, yeast, and bacteria have been sequenced, the structural similarities and

SUBSTITUTE SHEET differences of the malaria parasite to other G6PD can be easily identified. For instance, the reactive lysyl residue in the predicted binding site for glucose-6-phosphate were identical in mammalian (human and rat) , fruit fly, yeast, bacterial and parasite G6PD. The NADP binding site proposed by Beutler and colleagues based on convincing genetic evidence (A. Hirono, W. Kuhl, T. Gelbart, L. Forman, V.F. Fairbanks, E. Beutler, PNAS. 86, 10015-10017 (1989)) is not present in falciparum G6PD; however, the region proposed by Persson et al. based on recognizable characteristics of coenzyme binding sites, including GXXGXXA and β-α-β fold is present in the parasite deduced amino acid sequence. The surprising features of the predicted protein structure of the parasite G6PD enzyme, however, are its molecular mass, pi, and membrane associated motifs.

Pfg6pd, as compared to all of the other G6PD genes except E . coli that have been analyzed so far, has the least number of identical residues, and has a large insertion (residues 1-147) between the N-terminus and the putative NADP binding site and another large insertion (268-354) of 61 amino acids between that binding site and the G6P binding sites. These insertions make the predicted molecular mass of the monomer at least 82kDa rather than the 50-55kDa predicted for the other known G6PD enzymes. The N-terminal insertion contains two potentially important structures: a secretory signal sequence (residues 63-76) and a hydrophilic region (residues 123-135) . The other insertion contains a potential transmembrane helical structure (residues 349-364) that the other G6PD proteins lack, despite the identity of a number of residues in this region. Another membrane associated structure, a membrane spanning segment, is predicted toward the C-terminus (residues 614-630) . Finally, the remarkably slow migration of P. falciparum G6PD in native PAGE may be explained by its predicted higher molecular mass. Whether the unique features of P. falciparum G6PD target the enzyme to the endoplasmic reticulum for transport to the parasitophorous vacuole, or even to the RBC cytoplasm, or to another compartment within the parasite itself remain to be determined. Wherever the enzyme resides, the striking differences in the structure of G6PD between parasite and other organisms may potentially be exploited in the design of new chemotherapeutic agents against malaria.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Kaslow, David S. Q Shahabuddin, Mohammed 0

(ii) TITLE OF INVENTION: Isolation and Characterization of cDNA of Plasmodium Falciparum Glucose-6-Phosphate Dehydrogenase

(iii) NUMBER OF SEQUENCES: 2 O (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Cushman Darby & Cushman

(B) STREET: 1615 L St. N.W.

(C) CITY: Washington

(D) STATE: D.C.

(E) COUNTRY: U.S.A.

(F) ZIP: 20036-5601

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA: CO (A) APPLICATION NUMBER: US βj (B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

( (AA)) NNAAMMEE:: SSccootttt,, WWaattssoonn TT..

CO (B) REGISTRATION NUMBER: 26,581

I m (C) REFERENCE/DOCKET NUMBER: WTS/5683/92326/

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 861-3000

(B) TELEFAX: (202) 822-0944

(C) TELEX: 6714627 CUSH

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2750 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear O D (ii) MOLECULE TYPE: DNA (genomic)

( i) SEQUENCE DESCRIPTION: SEQ ID NO:l: ^m O

ATTTCCATAT TGCCAGTTTA TTTCCAAATA TATTTTATAA TATATATATG AATAACTATC 60 ι AAAATAATTA TATATATAAT GAAAAAACAT TAGATTTTAT AAATAATGAT CAAGATAATG 120

ATAATTTAAA ATATTTGAAA GAATATGTAT ATTTTACGAC AACAAATCAA TTTGATGTTA 180

GGAAAAGAAT TACAGTATCT TTAAATTT T TAGCTAATGC ATCAAGTAAA ATATTTTTAT 240

TAAATTCTAA AGACAAATTA GATTTATGGA AAAATATGTT GATTAAATCA TATATTGAAG 300

TGAATTATAA TTTATATCCA GCTACTTATT TAATAGATAC ATCATGCACC AACGAAAATG 360

TTAATATTAA CAATAACAAC AATAATAATA ATAAGAATAA GAATAATTAT TGTTATAGTA 420

ATACCACTGT TATATCTTGT GGTTATGAAA ATTATACAAA ATATATTGAA GAAATTTATG 480

CO c oo ATTCTAAATA TGCTCTATCT CTTTATTCTA ATAGTTTGAA TAAAGAAGAA TTATTAACTA 540

TAATAATTTT TGGCTGTTCA GGTGATTTAG CCAAAAAAAA AATATATCCA GCTTTATTTA 600

AATTATTTTG TAATAATTCC TTACCAAAAG ATTTATTAAT CATTGGATTT GCTAGAACAG 660 ^

CO

I fTl TTCAAGATTT CGATACATTT TTTGATAAAA TAGTTATATA TTTAAAACGA TGTTTATTAT 720

GTTATGAAGA TTGGTCTATA TCAAAAAAGA AGGATCTTTT AAATGGTTTT AAAAATAGGT 780

GTCGATATTT TGTTGGTAAT TATTCGTCTT CAGAAAGTTT TGAAAATTTT AATAAATATT 840

TAACAACTAT TGAAGAAGAA GAAGCAAAAA AAAAATATTA TGCAACATGT TATAAAATGA 900

ATGGTTCAGA TTATAATATA TCAAATAATG TTGCAGAGGA TAATATTAGT ATAGATGATG 960

AAAATAAGAC AAATGAATAT TTTCAAATGT GTACTCCAAA AAATTGCCCT GAT ATGTAT 1020

TTTCATCAAA TTATAATTTT CCATATGTTA TAAATAGTAT ATTATATTTA GCATTACCTC 1080

CACATATATT TATTAGTACT TTAAAAAAAA TTA AAAAA AAATTGTTTA AATAGTAAAG 1140

GCACTGATAA AA ATTACTA GAAAAACCAT TTGGAAATGA TTTAGATTCA TTTAAAATGT 1200

TATCAAAACA AATATTAGAG AATTTTAATG AACAACAAAT ATATAGAATA GATCATTATT 1260

H oo

TGGGTAAGGA TATGGTTTCA GGATTGTTGA AATTAAAATT TACAAATACA TTTTTATTAT 1320 O

CTTTAATGAA TAGACATTTT ATAAAATGTA TTAAAATTAC TCTTAAAGAA ACTAAAGGTG 1380

TATATGGTAG AGGACAATAT TTTGATCCCT ATGGTATTAT TAGAGATGTT ATGCAAAATC 1440

ATATGTTACA ATTATTAACA TTAATAACTA TGGAAGATCC TA AGATTTA AATGATGAAT 1500

CTGTAAAAAA TGAGAAAATA AAAATTCTTA AATCAATTCC TTCGATCAAA TTAGAAGATA 1560

CTATTATTGG ACAATATGAA AAAGCTGAAA ATTTTAAAGA AGATGAAAAT AATGATGATG 1620

AATCGAAAAA AAATCATAGT TATCATGATG ATCCACATAT AGATAAAAAT TCGATTACTC 1680

CAACATTTTG TACATGTATC TTATATATTA ATTCAATTAA TTGGTATGGT GTACCAATCA 1740

TTTTTAAATC TGGAAAAGGT CTGAATAAAG ATATATGTGA AATACGTATA CAATTCCATA 1800 O 0 ATAT ATGGG GTCGTCTGAT GAAAATATGA ATAATAATGA ATTTGTTATT ATATTACAAC 1860

CTGTTGAAGC TATATACCTA AAAATGATGA TTAAAAAAAC GGGTTGTGAA GAAATGGAAG 1920

__

AAGTACAATT AAACCTAACA GTGAATGAGA AAAATAAAAA AATTAATGTA CCAGAAGCAT 1980

CO

I πi ATGAAACATT ACTCTTAGAA TGTTTTAAAG GACATAAAAA AAAATTCATC TCAGACGAGG 2040 1 AATTGTATGA ATCATGGAGA ATATTTACTC CTTTACTTAA GGAACTCCAG GAAAAACAAG 2100

TCAAGCCTCT TAAATATTCT TTTGGATCAT CAGGCCCTAA AGAGGTATTT GGACTTGTCA 2160

AAAAATATTA CAATTATGGT AAAAATTATA CGCACAGACC TGAGTTTGTT AGAAAATCCT 2220

CTTTTTATGA AGACGATTTG TTAGATATTA ATTATTAATT GATATATGTA TATATTTAAA 2280

TTAACCAAAT TAACACCCAA TGAATATGAA AATAATATAT ATATATATAT ATATATTATA 2340

TGATTGTTTA GTATATTATT ACCTATCTTT TATAAGATAA CATAAATGTA TATATTATGA 2400

CATATATATA TA ATATATA TATTATTTCA CTTATCTGCC CACGAACTTT ATTTTTGTTT 2460

TAAAATTC A GTATAATTAA ATAAAAGAAA ATATTTGGAA CAATTTGCAT TTTTTATGTA 2520

TAAATAAAAT TTATATAATA ATATACTTTC ATACTTACTT TTTATTTTAT TTTATTTTAT 2580 o TTATTTTTTA AATGTCTATT ATAT TACAT A AAATGCGT TTTCAAATAA AT ATAAAAA 2640 O D CCCATGTTTA ACTAATAATA TTACAAATAG AACTCAAAAA AAAAAAAAAT AATTATACAA 2700 1

TGAATTAAAG CTTTTTAATA TATTTTTAAT GGTATCTCCA GACTTTAGAG 2750

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 751 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

_{_ 20 25 30 m

__ι lie Asn Asn Asp Gin Asp Asn Asp Asn Leu Lys Tyr Leu Lys Glu Tyr

35 40 45

Val Tyr Phe Thr Thr Thr Asn Gin Phe Asp Val Arg Lys Arg lie Thr 50 55 60

Val Ser Leu Asn Leu Leu Ala Asn Ala Ser Ser Lys lie Phe Leu Leu

Asn Ser Lys Asp Lys Leu Asp Leu Trp Lys Asn Met Leu lie Lys Ser

85 90 95

Tyr lie Glu Val Asn Tyr Asn Leu Tyr Pro Ala Thr Tyr Leu lie Asp 100 105 110

Thr Ser Cys Thr Asn Glu Asn Val Asn lie Asn Asn Asn Asn Asn Asn 1 115 120 125

Asn Asn Lys Asn Lys Asn Asn Tyr Cys Tyr Ser Asn Thr Thr Val lie t 130 135 140 O

Ser Cys Gly Tyr Glu Asn Tyr Thr Lys Tyr lie Glu Glu lie Tyr Asp ι 145 150 155 160

Ser Lys Tyr Ala Leu Ser Leu Tyr Ser Asn Ser Leu Asn Lys Glu Glu

165 170 175

Leu Leu Thr lie lie lie Phe Gly Cys Ser Gly Asp Leu Ala Lys Lys

Lys lie Tyr Pro Ala Leu Phe Lys Leu Phe Cys Asn Asn Ser Leu Pro 195 200 205

Lys Asp Leu Leu lie lie Gly Phe Ala Arg Thr Val Gin Asp Phe Asp 210 215 220

CO

I Lys Asn Arg Cys Arg Tyr Phe Val Gly Asn Tyr Ser Ser Ser Glu Ser m 260 265 270

Phe Glu Asn Phe Asn Lys Tyr Leu Thr Thr lie Glu Glu Glu Glu Ala 275 280 285

Lys Lys Lys Tyr Tyr Ala Thr Cys Tyr Lys Met Asn Gly Ser Asp Tyr 290 295 300

Asn lie Ser Asn Asn Val Ala Glu Asp Asn lie Ser lie Asp Asp Glu 305 310 315 320

Asn Lys Thr Asn Glu Tyr Phe Gin Met Cys Thr Pro Lys Asn Cys Pro

325 330 335

Asp Asn Val Phe Ser Ser Asn Tyr Asn Phe Pro Tyr Val lie Asn Ser 340 345 350 1 lie Leu Tyr Leu Ala Leu Pro Pro His lie Phe lie Ser Thr Leu Lys 355 360 365 t

Lys lie lie Lys Lys Asn Cys Leu Asn Ser Lys Gly Thr Asp Lys lie 370 375 380

Leu Leu Glu Lys Pro Phe Gly Asn Asp Leu Asp Ser Phe Lys Met Leu 385 390 395 400

Ser Lys Gin lie Leu Glu Asn Phe Asn Glu Gin Gin lie Tyr Arg lie

405 410 415

Asp His Tyr Leu Gly Lys Asp Met. Val Ser Gly Leu Leu Lys Leu Lys 420 425 430

Phe Thr Asn Thr Phe Leu Leu Ser Leu Met Asn Arg His Phe lie Lys 435 440 445

CO c Cys lie Lys lie Thr Leu Lys Glu Thr Lys Gly Val Tyr Gly Arg Gly

CD 450 455 460

21

Gin Tyr Phe Asp Pro Tyr Gly lie lie Arg Asp Val Met Gin Asn His 465 470 475 480 to LΠ

CO

I Met Leu Gin Leu Leu Thr Leu lie Thr Met Glu Asp Pro lie Asp Leu m 485 490 495 m

H

Asn Asp Glu Ser Val Lys Asn Glu Lys lie Lys lie Leu Lys Ser lie 500 505 510

Pro Ser lie Lys Leu Glu Asp Thr lie lie Gly Gin Tyr Glu Lys Ala 515 520 525

Glu Asn Phe Lys Glu Asp Glu Asn Asn Asp Asp Glu Ser Lys Lys Asn 530 535 540

His Ser Tyr His Asp Asp Pro His He Asp Lys Asn Ser He Thr Pro 545 550 555 560

Thr Phe Cys Thr Cys He Leu Tyr He Asn Ser He Asn Trp Tyr Gly D 565 570 575 }

Val Pro He He Phe Lys Ser Gly Lys Gly Leu Asn Lys Asp He Cys 580 585 590 t O Glu He Arg He Gin Phe His Asn He Met Gly Ser Ser Asp Glu Asn 595 600 605 ι Met Asn Asn Asn Glu Phe Val He He Leu Gin Pro Val Glu Ala He 610 615 620

Tyr Leu Lys Met Met He Lys Lys Thr Gly Cys Glu Glu Met Glu Glu 625 630 635 640

Val Gin Leu Asn Leu Thr Val Asn Glu Lys Asn Lys Lys He Asn Val

645 650 655

Pro Glu Ala Tyr Glu Thr Leu Leu Leu Glu Cys Phe Lys Gly His Lys 660 665 670

CO c Lys Lys Phe He Ser Asp Glu Glu Leu Tyr Glu Ser Trp Arg He Phe

CD 675 680 685

23

Thr Pro Leu Leu Lys Glu Leu Gin Glu Lys Gin Val Lys Pro Leu Lys 690 695 700 ro

CO Tyr Ser Phe Gly Ser Ser Gly Pro Lys Glu Val Phe Gly Leu Val Lys m 705 710 715 720

Lys Tyr Tyr Asn Tyr Gly Lys Asn Tyr Thr His Arg Pro Glu Phe Val

725 730 735

Arg Lys Ser Ser Phe Tyr Glu Asp Asp Leu Leu Asp He Asn Tyr 740 745 750

Claims

WHAT IS CLAIMED IS:

1. A purified DNA segment, wherein said segment has a nucleotide sequence or a unique portion of said sequence as shown in Fig. 1 (SEQ ID NO:l) .

2. A protein, wherein said protein has an amino acid sequence or a unique portion of said sequence as shown in Fig. 2 (SEQ ID NO:2) .

3. A DNA segment encoding the protein of claim 2.

4. The protein according to claim 2 separated from proteins with which said protein is naturally associated.

5. A reco binantly produced protein having at least a unique portion of the amino acid sequence given in Fig. 2 (SEQ ID NO:2).

6. A recombinant DNA molecule comprising: i) said DNA segment according to claim 3; and ii) a vector.

7. A host cell stably transfected with the recombinant DNA molecule of claim 6 in a manner allowing expression of a functionally active form of said protein encoded by said DNA molecule.

8. The host cell according to claim 7 which is Eεcherichia coli.

9. The host cell according to claim 7 which is a eukaryotic cell.

10. A method of producing a recombinant Plaεmodium falciparum glucose-6-phosphate dehydrogenase protein comprising culturing said host cells according to claim 7, in a manner allowing expression of said protein and isolation of said protein.

11. A method of screening drugs for activity against Plasmodium falciparum glucose-6- phosphate dehydrogenase comprising the steps of: i) contacting said drug to the host cell of claim 7,

TE SHEET ii) placing said drug-contacted host cell into an environment wherein all glucose is labelled glucose, iii) detecting the presence or absence of a labelled reaction product of said labelled glucose and Plasmodium falciparum glucose-6-phosphate dehydrogenase; and iv) performing appropriate control assays.

12. An antibody specific for the protein encoded by said DNA segment according to claim 1.

13. The antibody according to claim 12 which is polyclonal.

14. The antibody according to claim 12 which is monoclonal.

15. A bioassay for the diagnosis of P. falciparum infection comprising the steps of: i) coating a surface with antibodies according to claim 12; ii) contacting said coated surface with serum from a mammal suspected of infection with P. falciparum; and iii) detecting the presence or absence of a complex formed between said antibodies and proteins present in the serum.

16. A vaccine against malaria comprising all, or a unique portion of a protein encoded by said DNA segment according to claim 1, in an amount sufficient to induce immunization against said disease, and a pharmaceutical carrier.

17. The vaccine according to claim 16 which further comprises an adjuvant.

SUBSTITUTE SHEET