WO1999055848A2 - Clonage et expression de plasmepsines recombinantes - Google Patents

Clonage et expression de plasmepsines recombinantes Download PDF

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Publication number
WO1999055848A2
WO1999055848A2 PCT/US1999/009548 US9909548W WO9955848A2 WO 1999055848 A2 WO1999055848 A2 WO 1999055848A2 US 9909548 W US9909548 W US 9909548W WO 9955848 A2 WO9955848 A2 WO 9955848A2
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Prior art keywords
plasmepsin
lys
amino acid
phe
leu
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PCT/US1999/009548
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English (en)
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WO1999055848A3 (fr
Inventor
John Barton Dame
Charles Adam Yowell, Jr.
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University Of Florida
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Priority to AU37794/99A priority Critical patent/AU3779499A/en
Publication of WO1999055848A2 publication Critical patent/WO1999055848A2/fr
Publication of WO1999055848A3 publication Critical patent/WO1999055848A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the malaria parasite spends part of its life cycle within erythrocytes.
  • parasites break down hemoglobin to obtain amino acids for protein synthesis.
  • Goldberg and colleagues Goldberg et al, 1990; Goldberg 1992 have described the pathway of hemoglobin degradation in the malaria parasite P. falciparum .
  • Plasmodium Aspartic proteinases of the human malaria parasite, Plasmodium falciparum, are the best studied (Vander Jagt et al, 1987; Vander Jagt et al, 1989; Goldberg et al, 1991; Gluzman et al, 1994; Francis et al, 1994; Symbol et al, 1994), and multiple forms of this enzyme have been identified (Vander Jagt et al, 1992; Gluzman et al, 1994). Two forms have been characterized in the food vacuole of the asexual, bloodstage parasite (Vander Jagt et al, 1992). These enzymes from P.
  • Plasmodium spp. express multiple, distinct plasmepsins is unknown.
  • Plasmepsins examined from Plasmodium species infecting man are single polypeptide chains of
  • the active, mature plasmepsins are 327-329 amino acids in length with an isoelectric point of 4.25-4.67, as determined from amino acid sequence analysis.
  • Native plasmepsins have been prepared only from P. falciparum (Vander Jagt et al, 1992; Gluzman et al, 1994).
  • the genes encoding the mature plasmepsin, plus 48 amino acids of the proregion, have been engineered for cloning into the BamHI site of the pET3a cloning vector (Hill et al, 1994).
  • Recombinant proplasmepsins were recovered from inclusion bodies, solubilized in urea, and refolded, as described (Hill et al, 1994). Purification is accomplished via ammonium sulfate precipitation and/or ion exchange chromatography, and self- activation is accomplished by brief pre-incubation under optimal assay conditions in the absence of substrate (Hill et al, 1994).
  • Plasmepsins from all species of the malaria parasite are believed to function as hemoglobinases, digesting the contents of the erythrocyte in which they develop. Although this has only been demonstrated directly for the plasmepsins of P. falciparum, the common life histories of the malaria parasites suggests that this function is shared by the plasmepsins of the other species. P. falciparum is not closely related evolutionarily to the other three species infectious for humans (Waters et al, 1991; Escalante et al, 1995).
  • Plasmepsin has a long proregion (123-124 amino acids) as compared to most other aspartic proteinases (ca. 50 amino acids) (Francis et al, 1994; Dame et al, 1994).
  • a novel proteolytic enzyme activity has been identified that activates the proenzyme by removing this proregion of the P. falciparum plasmepsins.
  • Self-activated, recombinant plasmepsins have from 2 to 12 additional amino acids at the N-terminus (Hill et al, 1994).
  • Human cathepsin D is the peptidase of the human host for the malaria parasite that is most closely similar to the plasmepsins in structure and function. Inhibitors which may eventually be used as antimalarial drugs must differentially inhibit plasmepsins versus this important host enzyme.
  • the subject invention concerns cloning and expression of genes encoding plasmepsins from Plasmodium, the parasite responsible for malaria in humans.
  • One aspect of the invention pertains to polynucleotide sequences that encode active plasmepsin enzymes. Specifically described are polynucleotides encoding plasmepsins from P. vivax, P. ovale , and P. malariae.
  • the subject invention also concerns plasmepsin polypeptides encoded by the polynucleotides of the invention.
  • the plasmepsin polypeptides of the invention possess activity as aspartic proteinases and can be used to screen for drugs which block or inhibit the activity of the plasmepsins of the present invention.
  • Figure 1 shows a polynucleotide sequence encoding a plasmepsin of P. vivax according to the present invention.
  • Figure 2 shows amino acid sequence in standard single-letter amino acid symbols of a plasmepsin of P. vivax encoded by the sequence shown in Figure 1.
  • Figure 3 shows a polynucleotide sequence encoding a plasmepsin of P. ovale according to the present invention.
  • Figure 4 shows an amino acid sequence in standard single-letter amino acid symbols of a plasmepsin of P. ovale encoded by the sequence shown in Figure 3.
  • Figure 5 shows a polynucleotide sequence encoding a plasmepsin of P. malariae according to the present invention.
  • Figure 6 shows an amino acid sequence in standard single-letter amino acid symbols of a plasmepsin of P. malariae encoded by the sequence shown in Figure 5.
  • the subject invention concerns cloning and expression of recombinant plasmepsins from Plasmodium species.
  • One aspect of the present invention concerns polynucleotide molecules that encode active plasmepsin enzymes from Plasmodium species, including P. vivax, P. ovale and P. malariae.
  • Polynucleotides of the invention can be used to express the plasmepsin enzymes.
  • Polynucleotide molecules of the present invention can be prepared using standard materials and methods known in the art, such as, for example, PCR amplification using suitable primers.
  • the invention also concerns plasmepsin polypeptides encoded by the polynucleotides of the invention.
  • the purified polypeptides can be obtained by expression of recombinant vectors comprising polynucleotide sequences of the invention.
  • Plasmepsin protein can be purified according to the methods disclosed herein.
  • the active plasmepsins of the invention function as aspartic proteinases.
  • the subject plasmepsins can also be used to screen for drugs or other compounds or molecules that can inhibit or block the activity of the plasmepsin enzymes.
  • the subject invention also pertains to methods for screening for drugs that are active against one or more species of Plasmodium by testing for inhibitory action of a drug or compound against a plasmepsin of the present invention.
  • the subject invention also pertains to polynucleotides that are antisense to DNA encoding the subject plasmepsins, or RNA transcribed from plasmepsin encoding DNA.
  • the subject invention also concerns processes for preparing plasmepsin proteins of the invention.
  • One embodiment of the invention comprises expressing a polynucleotide of the invention that encodes a plasmepsin protein.
  • the polynucleotide is provided on a suitable expression vector comprising regulatory sequences, such as promoters, polyadenylation signal, and the like.
  • the expression vector is delivered into a host cell and the subject polynucleotide expressed to produce a plasmepsin expression product.
  • the plasmepsin produced can then be purified from the cell.
  • the subject invention also concerns antibodies that bind to plasmepsin polypeptides of the invention. These antibodies can be monoclonal or polyclonal in nature.
  • the antibodies are monoclonal antibodies.
  • Antibodies that bind to a plasmepsin from a single species of Plasmodium are specifically contemplated.
  • Antibodies that bind to plasmepsin from more than one species of Plasmodium are also within the scope of the invention.
  • Antibodies of the subject invention can be prepared using standard materials and methods known in the art (see, for example, Monoclonal Antibodies: Principles and Practice, 1983; Monoclonal Hybridoma Antibodies: Techniques and Applications, 1982; Selected Methods in Cellular Immunology, 1980; Immunological Methods, Vol. II, 1981; Practical Immunology, and Kohler et al, 1975).
  • the subject invention also concerns oligonucleotide primers and probes that can hybridize to a polynucleotide of the subject invention.
  • the oligonucleotide primers and probes can be used to detect or identify Plasmodium species.
  • the subject invention also pertains to vectors comprising polynucleotide sequences of the present invention.
  • vectors include, for example, cloning vectors, expression vectors and the like. Numerous vectors which can be used according to the present invention are known in the art.
  • cells, microorganisms, viruses and the like that comprise the polynucleotides and/or polypeptides of the invention.
  • the cells can be either eucaryotic or prokaryotic cells.
  • Prokaryotic cells include, for example, E coli., Bacillus species and others.
  • Eucaryotic cells include, for example, yeast cells, insect cells, and mammalian cells.
  • Microorganisms and cells comprising polynucleotides of the invention can be used to express sufficient quantities of the plasmepsin for purification purposes.
  • any of a number of different nucleotide sequences can be used, based on the degeneracy of the genetic code, to produce the plasmepsin proteins described herein. Accordingly, any nucleotide sequence which encodes the plasmepsin proteins described herein comes within the scope of this invention and the claims appended hereto. Also, as described herein, fragments of the plasmepsin proteins are an aspect of the subject invention so long as such fragments retain the biological activity so that such fragments are useful in screening, therapeutic and/or diagnostic procedures as described herein. Such fragments can easily and routinely be produced by techniques well known in the art. For example, time-controlled
  • BaB 1 exonuclease digestion of the full-length DNA followed by expression of the resulting fragments and routine screening methods can be used to readily identify expression products having the desired activity (Wei et al, 1983).
  • nucleic acid and “polynucleotide sequence” refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double- stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally-occurring nucleotides.
  • the polynucleotide sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein.
  • the polynucleotide sequences include both full-length sequences as well as shorter sequences derived from the full-length sequences.
  • polynucleotide sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell. Allelic variations of the exemplified sequences also come within the scope of the subject invention.
  • the polynucleotide sequences falling within the scope of the subject invention further include sequences which specifically hybridize with the exemplified sequences under stringent conditions.
  • the polynucleotide includes both the sense and antisense strands as either individual strands or in the duplex.
  • polynucleotides and polypeptides of the subject invention also encompass variant sequences containing mutations in the exemplified sequences.
  • These mutations in nucleotide or amino acid sequences of the polynucleotides and polypeptides of the invention, respectively, can include, for example, nucleotide or amino acid substitutions, insertions, and deletions as long as the variant sequence has substantial sequence identity with an exemplified sequence of the present invention.
  • this sequence identity is greater than about 50%; more preferably, the sequence identity is greater than about
  • sequence identity refers to the homology between two distinct sequences as determined using, for example, the GCG FASTA software program (University of Wisconsin).
  • hybridize or “hybridizing” refer to the binding of two single-stranded nucleic acids via complementary base pairing.
  • hybridizing specifically to refers to binding, duplexing, or hybridizing of a molecule to a nucleotide sequence under stringent conditions when that sequence is present in a preparation of total cellular DNA or RNA.
  • stringent conditions refers to conditions under which a polynucleotide molecule, such as a DNA probe, will hybridize to its target sub-sequence, but not to sequences having little or no homology to the target sequence.
  • stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a complementary probe.
  • stringent conditions will be those in which the salt concentration is at least about 0.1 to 1.0 N Na ion concentration at a pH of about 7.0 to 7.5 and the temperature is at least about 60°C for long sequences (e.g., greater than about 50 nucleotides) and at least about 42 °C for shorter sequences (e.g., about 10 to 50 nucleotides).
  • isolated or substantially pure when referring to polynucleotide sequences encoding the plasepsin proteins or fragments thereof refers to polynucleotides which encode plasmepsin proteins or peptides and which are no longer in the presence of sequences with which they are associated in nature.
  • isolated or “substantially purified” when referring to the plasmepsin proteins of the subject invention means a chemical composition which is essentially free of other cellular components. It is preferably in a homogenous state and can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • a protein which is the predominant species present in a preparation is substantially purified. Generally, a substantially purified or isolated protein will comprise more than 80%> of all macromolecular species present in the preparation. Preferably, the protein is purified to represent greater than 90% of all macromolecular species present. More preferably, the protein is purified to greater than 95%, and most preferably the protein is purified to essential homogeneity, wherein other macromolecular species are not detected by conventional techniques.
  • PCR Polymerase Chain Reaction
  • thermostable DNA polymerase such as Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated.
  • the DNA sequences of the subject invention can be used as primers for PCR amplification.
  • a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the 5' end) of the exemplified primers fall within the scope of the subject invention. Mutations, insertions and deletions can be produced in a given primer by methods known to an ordinarily skilled artisan. It is important to note that the mutational, insertional, and deletional variants generated from a given primer sequence may be more or less efficient than the original sequences. Notwithstanding such differences in efficiency, these variants are within the scope of the present invention.
  • PCR-amplified DNA may serve as a hybridization probe.
  • the nucleotide sequences of the subject invention as probes, the
  • DNA can first be obtained in its native, double-stranded form.
  • a number of procedures are currently used to isolate DNA and are well known to those skilled in this art.
  • One approach for the use of the subject invention as probes entails first identifying by Southern blot analysis of a DNA library all DNA segments homologous with the disclosed nucleotide sequences. Thus, it is possible, without the aid of biological analysis, to know in advance the presence of genes homologous with the polynucleotide sequences described herein. Such a probe analysis provides a rapid diagnostic method.
  • One hybridization procedure useful according to the subject invention typically includes the initial steps of isolating the DNA sample of interest and purifying it chemically. For example, total fractionated nucleic acid isolated from a biological sample can be used. Cells can be treated using known techniques to liberate their DNA
  • the DNA sample can be cut into pieces with an appropriate restriction enzyme.
  • the pieces can be separated by size through electrophoresis in a gel, usually agarose or acrylamide.
  • the pieces of interest can be transferred to an immobilizing membrane in a manner that retains the geometry of the pieces.
  • the membrane can then be dried and prehybridized to equilibrate it for later immersion in a hybridization solution.
  • the manner in which the nucleic acid is affixed to a solid support may vary. This fixing of the DNA for later processing has great value for the use of this technique in field studies, remote from laboratory facilities.
  • nucleotide segments of the subject invention which are used as probes can be synthesized by use of DNA synthesizers using standard procedures.
  • the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels.
  • Typical radioactive labels include 32 P, 35 S, or the like.
  • a probe labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by a conventional nick translation reaction, using a DNase and DNA polymerase.
  • probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting.
  • enzymes such as polynucleotide kinase or terminal transferase to end-label the DNA for use as probes.
  • Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or perixodases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives.
  • the probes may be made inherently fluorescent as described in International Application No. WO93/16094.
  • the probe may also be labeled at both ends with different types of labels for ease of separation, as, for example, by using an isotopic label at the end mentioned above and a biotin label at the other end.
  • the amount of labeled probe which is present in the hybridization solution will vary widely, depending upon the nature of the label, the amount of the labeled probe which can reasonably bind to the filter, and the stringency of the hybridization. Generally, substantial excesses of the probe will be employed to enhance the rate of binding of the probe to the fixed DNA.
  • hybridization is conducted under stringent conditions by techniques well known in the art, as described, for example, in Keller and Manak, 1987.
  • nucleotide sequences of the subject invention include mutations
  • Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.
  • the known methods include, but are not limited to:
  • mutational, insertional, and deletional variants generated from a given probe may be more or less efficient than the original probe. Notwithstanding such differences in efficiency, these variants are within the scope of the present invention.
  • the amino acid sequence of a protein is determined by the nucleotide sequence of the DNA. Because of the redundancy of the genetic code, i.e., more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleotide sequences can code for a particular amino acid.
  • the amino acid sequence of the proteins of the subject invention can be encoded by equivalent nucleotide sequences encoding the same amino acid sequence of the protein. Accordingly, the subject invention also includes probes which would hybridize with various polynucleotide sequences which would all code for a given protein or variations of a given protein.
  • proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser and Kezdy, 1984).
  • Recombinant plasmepsins were cloned from genomic DNA by PCR amplification (Reddy et al, 1993).
  • the enzymes were expressed using a pET3a (Novagen) expression plasmid transformed into BL21 (DE3) pLysS Escherichia coli (Stratagene).
  • the protein was isolated as inclusion bodies and solubilized, refolded, and purified using the methods described by Hill et al. (1994) with slight variations (Westling et al, 1997). Briefly, the inclusion bodies were solubilized in 8 M urea, 0.05 M CAPS, pH 10.5, 0.005 EDTA, 0.2 M 2-mercaptoethanol at a final concentration of 1 mg/ml.
  • the protein was allowed to refold by dialysis against 13 times the volume of 20 m Tris, pH 8, buffer at 4°C.
  • the buffer was changed every 6 hr for a total of three changes.
  • the refolded protein was purified by anion exchange chromatography using a Resource Q column (Pharmacia).
  • the dialysate was loaded onto the column in 20 mM Tris buffer, pH 8, at 4°C and the protein was eluted using a gradient of 20 m Tris, 1 M NaCl, pH 8, buffer.
  • a peak of active protein was obtained at 180 mM NaCl and was homogenous by SDS- PAGE analysis. Edman degradation was performed to confirm the N-terminal sequence of the protein obtained (Protein Chemistry Core Facility, University of Florida).
  • Vander Jagt D.L., W.S. Caughey, N.M. Campos, L.A. Hunsaker, M.A. Zanner (1989) "Parasite proteases and antimalarial activities of protease inhibitors" Prog. Clin. Biol. Res. 313:105-118. Vander Jagt, D.L., L.A. Hunsaker, N.M. Campos (1987) "Comparison of proteases from chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum” Biochem. Pharmacol. 36:3285-3291.
  • CTCCCTGTAC ATGACGTCCA CGCTGGTTAC CTAACCATTG GTGGAATTGA AGAGAAGTTT 900
  • Gly Ser lie Asp Pro lie Val Val Glu Leu Lys Asn Gin Asn Lys lie 260 265 270
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO : 4 :
  • MOLECULE TYPE DNA (genomic)
  • AATTATTTCT TTTCCCCAAA TTATAAAGTG AATAAAATTG TACAAAATAC GGAACATTTA 240

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Abstract

La présente invention concerne le clonage et l'expression de plasmepsines provenant de Plasmodium. Dans l'un de ses aspects, l'invention se rapporte à des séquences polynucléotidiques qui codent les enzymes plasmepsines actives, et elle se rapporte en particulier à des polynucléotides codant les plasmepsines de P. vivax, P. ovale et P. malariae. L'invention concerne aussi les polypeptides de la plasmepsine codés par les polynucléotides de l'invention. Les polynucléotides de l'invention, qui possèdent une activité de protéinases aspartiques, peuvent être utilisés pour cribler des médicaments qui bloquent ou inhibent l'activité des plasmepsines de l'invention.
PCT/US1999/009548 1998-04-30 1999-04-30 Clonage et expression de plasmepsines recombinantes WO1999055848A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU37794/99A AU3779499A (en) 1998-04-30 1999-04-30 Cloning and expression of recombinant plasmepsins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US7033798A 1998-04-30 1998-04-30
US09/070,337 1998-04-30

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WO1999055848A2 true WO1999055848A2 (fr) 1999-11-04
WO1999055848A3 WO1999055848A3 (fr) 2000-04-13

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DAME, J.B. ET AL: "Sequence, expression and modeled structure of an aspartic proteinase from the human malaria parasite Plasmodium falciparum" MOLECULAR AND BIOCHEMICAL PARASITOLOGY, vol. 64, 1994, pages 177-190, XP002120909 cited in the application *
DATABASE EMBL/GENBANK/DDBJ [Online] Accession No. AF001209 5 May 1998 (1998-05-05) DAME, J.B. ET AL.: "Cloning and expression of plasmepsins from the human malaria parasites Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae" XP002120910 *
DATABASE EMBL/GENBANK/DDBJ [Online] Accession No. AJ223308, 4 September 1998 (1998-09-04) HUMPHREYS, M.J.: "Cloning and expression of a gene encoding the proplasmepsin from plasmodium berghei" XP002120911 *
FRANCIS, S.E. ET AL.: "Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase" EMBO JOURNAL, vol. 13, no. 2, 1994, pages 306-317, XP002120908 cited in the application *
WESTLING, J. ET AL.: "Plasmodium falciparum., P. vivax, and P. malariae: A comparison of the active sites properties of plasmepsins cloned and expressed from three different species of the malariae parasite" EXPERIMENTAL PARASITOLOGY, vol. 87, no. 3, November 1997 (1997-11), pages 185-193, XP002121906 *

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