# Patent

## Title
Nucleic acid molecule encoding a cystine knot polypeptide

## Abstract

The present invention relates to a novel nucleic acid molecule encoding an amino acid sequence, which is capable of forming a cyclic structure. Cyclization may occur within a cell or cell membrane, or linear forms of the molecules may be circularised or partially circularised, in vitro using isolated enzyme systems or chemical means. The cyclised amino acid sequence is generally in the form of a stabilized folded structure such as acyclic knotted peptide, polypeptide or protein or functional equivalent. The nucleic acid molecules and cyclic and linear peptides are useful inter alia in the generation of molecules having animal or plant therapeutic properties, as well as in a range of diagnostic, industrial and agricultural, including horticultural, applications. Of particular importance is the use of these molecules in the protection of plants, such as crop plants, from pest and/or pathogen infestation.

## Background
This application is a Continuation of U.S. application Ser. No. 10/129,201, filed Sep. 10, 2002, issued as U.S. Pat. No. 7,592,433 on Sep. 22, 2009, which is a 371 US National Entry of PCT/AU2000/01352, filed Nov. 3, 2000, which claims the benefit of Australian Application Nos.: PQ3884, filed Nov. 5, 1999, and PQ4235, filed Nov. 25, 1999, each of which are incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates generally to a novel nucleic acid molecule encoding an amino acid sequence wherein said amino acid sequence or a derivative form thereof is capable of forming a cyclic structure. Cyclization may occur, for example, within a cell or cell membrane or linear forms of the molecules may be circularized or at least partly circularized in vitro using, for example, isolated enzyme systems or chemical means. The cyclised amino acid sequence is generally in the form of a stabilized folded structure such as a cyclic knotted peptide, polypeptide or protein or its functional equivalent. The present invention is further directed to cyclized molecules and in particular cyclic peptides, polypeptides or proteins, linear forms thereof including non-cyclic structural homologues of the cyclic peptides, polypeptides and proteins and precursor or derivative forms thereof encoded by the subject nucleic acid molecules. The nucleic acid molecules and cyclic and linear peptides, polypeptides and proteins of the present invention are useful inter alia in the generation of molecules having animal or plant therapeutic properties as well as in a range of diagnostic, industrial and agricultural including horticultural applications. Of particular importance is the use of these molecules in the protection of plants such as crop plants from pest and/or pathogen infestation. The cyclic and linear peptides, polypeptides and polypeptides may be naturally occurring or may be modified by the insertion or substitution of heterologous amino acid sequences. The therapeutic properties may be inherent in the naturally occurring cyclic or linear molecules and/or may be associated with the heterologous amino acid sequence. The present invention further provides microbial, plant and animal cell systems as well as in vitro systems capable of cyclizing linear forms of the peptides, polypeptides and proteins of the present invention. The present invention also extends to the peptide, polypeptide or protein sequences which are capable of cyclizing in the absence of any other exogenous factor and more specifically capable of circularizing through a catalytic process being an inherent activity of the peptides, polypeptides or proteins.
BACKGROUND OF THE INVENTION
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
A number of macrocyclic peptides with diverse biological activities have been discovered in plants in the Rubiaceae and Violaceae families. These include kalata B1 (1), the circulins (2), cyclopsychotride (3) and several peptides from Viola species (4-6). They range in size from 29-31 amino acids and contain six conserved Cys residues. These macrocyclic peptides differ from classical proteins in that they have no free N- or C-terminus due to their amide-circularized backbone. They also incorporate a cystine knot in which an embedded ring in the structure formed by two disulfide bonds and their connecting backbone segments is threaded by a third disulfide bond. These combined features of the cyclic cystine knot (CCK) produce a unique protein fold that is topologically complex and has exceptional chemical and biological stability.
Small cyclic peptides are also known in nature, particularly as antibiotics of microbial origin, and appear to have advantages of improved stability and biological activity over their non-cyclic counterparts. Because of their favourable properties, cyclic peptides (or mimics of them) have had pharmaceutical applications. One example is the immunosuppressant, cyclosporine. These classical cyclic peptides invariably comprise fewer than 15 amino acids, usually lack disulfide bonds and generally do not have well defined three dimensional structures. Such peptides are not gene products but are thought to be biosynthesized, non-ribosomally, via peptide synthetases.
In work leading up to the present invention, the inventors investigated the genetic basis of the macrocyclic peptides. In contrast to small cyclic peptides, the macrocyclic peptides, referred to herein as “cyclotides”, are encoded for by gene sequences and exhibit folding structures characteristic of true proteins. The elucidation of the genetic basis behind the cyclotides enables their expression and manipulation in transgenic plant, animal and microbial cells. Being cyclic, the cyclotides have a range of potential therapeutic, diagnostic, industrial and agricultural including horticultural applications. The cyclizing enzyme or enzymes themselves also have utility in the development of in vivo or in vitro systems for cyclizing target peptides, polypeptides and proteins. Furthermore, the present invention permits the generation of linear structural homologues of peptides, polypeptides and proteins.
SUMMARY OF THE INVENTION
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Nucleotide and amino acid sequences are referred to by a sequence identifier, i.e. <400>1, <400>2, etc. A sequence listing is provided after the claims.
One aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell to form a cyclic backbone wherein said cyclic backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three dimensional structure of said backbone.
Another aspect of the present invention present is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell to form a cyclic knotted peptide, polypeptide or protein.
A further aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes an amino acid sequence capable of forming a cyclic backbone wherein the cyclic backbone comprises the structure:—




wherein

    
    
        C is cysteine;
        each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe] and [XV1 . . . XVf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and
        wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and each of a to f may be the same or different and range from 1 to about 20.
    
    


Still another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes an amino acid sequence capable of forming a cyclic backbone wherein the cyclic backbone comprises the structure:—




wherein

    
    
        C is cysteine;
        each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe] and [XV1 . . . XVf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and
        wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and each of a to f may be the same or different and range from 1 to about 10.
    
    


Even still another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes an amino acid sequence capable of forming a cyclic backbone wherein the cyclic backbone comprises the structure:—




wherein

    
    
        C is cysteine;
        each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe] and [XV1 . . . XVf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and
        wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and wherein a is from about 3 to about 6, b is from about 3 to about 5, c is from about 2 to about 7, d is about 1 to about 3, e is about 3 to about 6 and f is from about 4 to about 9.
    
    


Yet another aspect of the present invention provides a nucleic acid molecule an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell in an in vitro cyclizing system to form a cyclic backbone wherein the cyclic backbone comprises the structure:—




wherein

    
    
        C is cysteine;
        each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe] and [XV1 . . . XVf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and
        wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and each of a is about 3, b is about 4, c is about from 4 to about 7, d is about 1, e is about 4 or 5 and f is from about 4 to about 7.
    
    


Even yet another aspect of the present invention provides a nucleic acid molecule an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell in an in vitro cyclizing system to form a cyclic backbone wherein the cyclic backbone comprises the structure:—




wherein

    
    
        C is cysteine;
        each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe] and [XV1 . . . XVf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and
        wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and wherein a is about 6, b is about 5, c is about 3, d is about 1, e is about 5 and f is about 8.
    
    


A further aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of forming a structural homologue of a cyclic peptide, polypeptide or protein within a cell or a membrane of a cell to form a backbone wherein said backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three-dimensional structure of said backbone wherein said backbone comprises free amino and carboxy termini.
Another aspect of the present invention contemplates a method of identifying nucleic acid molecules which encode one or more enzymes required for cyclization of an amino acid sequence said method comprising obtaining a nucleic acid molecule which encodes a precursor form of an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein; introducing or fusing to said nucleic acid molecule, a nucleotide sequence which encodes a reporter molecule capable of providing a detectable signal wherein said nucleotide sequence is inserted or fused to a portion of the nucleic acid molecule which is cleaved off prior to or during cyclization; introducing said nucleic acid molecule comprising the nucleotide sequence encoding the reporter molecule into a bank of cells carrying a DNA library comprising all or part of genomic DNA or cDNA from a plant which carries the enzyme or enzymes required for cyclization of an amino acid sequence; screening for and selecting cells which do not synthesize the reporter molecule.
A further aspect of the present invention contemplates a genetically modified cell or cells or a plant or animal comprising said genetically modified cells, said cells comprising a nucleic acid molecule having a nucleotide sequence or complementary nucleotide sequence which encodes an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein.
Still another aspect of the present invention contemplates a method of incorporating an amino acid sequence conferring a particular trait into a cyclic peptide, polypeptide or protein, said method comprising fusing or introducing a nucleotide sequence encoding said amino acid sequence to or into a second nucleotide sequence wherein said second nucleotide sequence encodes a peptide, polypeptide or protein which peptide, polypeptide or protein or a derivative therefor is capable of being cyclized into a knotted peptide, polypeptide or protein.
Even still another aspect of the invention contemplates an isolated nucleic acid molecule comprising the following nucleotide sequence:—

    
    
        [X1 . . . Xb[n1n2 . . . na]y1 . . . yc]d 

wherein

        [n1n2 . . . na] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein; and
        X1 . . . Xb and y1 . . . yc represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d may be any number and when d is >1, the amino acid sequence may be unique for each integer of d.
    
    


Yet another aspect of the invention contemplates an isolated nucleic acid molecule comprising the following nucleotide sequence:—

    
    
        j1 . . . je[X1 . . . Xa[n1n2 . . . na]y1 . . . yc]dq1 . . . qf 

wherein

        [n1n2 . . . na] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein;
        X1 . . . Xb and y1 . . . yc represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d may be any number and when d is >1, the amino acid sequence may be unique for each integer of d; and
        j1 . . . je and q1 . . . qf represent nucleotide sequences encoding a peptide, polypeptide or protein capable of directing the peptide, polypeptide or protein to a cellular compartment or organelle where a, b, c, d, e and f may be any number, where d is >1, the amino acid sequence may be unique for each integer of d.
    
    


Even yet another aspect of the invention contemplates an isolated nucleic acid molecule comprising the following nucleotide sequence:—

    
    
        [X1 . . . Xb[n1n2 . . . (k1 . . . kδ)λna]y1 . . . yc]d 

wherein

        [n1n2 . . . na] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein;
        X1 . . . Xb and y1 . . . yc represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d, δ and λ may be any number and when d or λ is >1, the amino acid sequence may be unique for each integer of d and λ;
        k1 . . . kδ represent a nucleotide sequence encoding an amino acid sequence conferring a particular activity or other trait.
    
    


Another aspect of the invention contemplates an isolated nucleic acid molecule comprising the following nucleotide sequence:—

    
    
        [n1n2 . . . (n11n21 . . . nγ1) . . . na]m 

wherein

        [n1n2 . . . na] and (n11n21 . . . nγ1) represent polynucleotide sequences encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein; and
        γ and a and m may be any number and when m is >1, the amino acid sequence may be unique for each integer of m.
    
    


A further aspect of the invention contemplates an isolated nucleic acid molecule comprising the following nucleotide sequence:—

    
    
        j1 . . . je[X1 . . . Xb[n1n2 . . . (n11n21 . . . (k1 . . . kδ)λna1)mna]y1 . . . yc]dq1 . . . qf 

wherein

        [n1n2 . . . na] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein;
        X1 . . . Xb and y1 . . . yc represents a polynucleotide sequence capable of encoding an amino acid sequence where a and b and c and d and e may be any number and when d is >1, the amino acid sequence may be unique for each integer of d;
        j1 . . . je and q1 . . . qf represents a nucleotide sequence encoding a peptide, polypeptide or protein capable of directing the peptide, polypeptide or protein to a cellular compartment or organelle;
        k1 . . . kδ represents a nucleotide sequence encoding an amino acid sequence conferring a particular activity or other trait;
        λ and m and d may be any number and when λ and m and d are each >1, the amino acid sequence may be unique for each integer of λ, m and d.
    
    


Yet another aspect of the present invention further contemplates a genetically modified plant which comprises a nucleotide sequence which encodes an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein and which confers on said plant a trait not present in the same species or variety of plant prior to genetic modification.
Even yet another aspect of the present invention provides the use of a nucleic acid molecule encoding an amino acid sequence, which amino acid sequence or a derivative or precursor form thereof is capable of being cyclized into a knotted peptide, polypeptide or protein, in the manufacture of a transgenic or genetically modified plant capable of producing said cyclic knotted peptide, polypeptide or protein.
Still yet another aspect of the present invention relates to an immunointeractive molecule specific for a peptide, polypeptide or protein when in cyclic form and encoded by a nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell to form a cyclic backbone wherein said cyclic backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three dimensional structure of said backbone.
Another aspect of the present invention is directed to an immunointeractive molecule specific for a peptide, polypeptide or protein encoded by a nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of forming a structural homologue of a cyclic peptide, polypeptide or protein within a cell or a membrane of a cell to form a backbone wherein said backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three dimensional structure of said backbone wherein said backbone comprises free amino and carboxy termini.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a representation showing (A) the amino acid sequence of Kalata B1 (SEQ ID NO: 41). The Kalata B1 peptide is composed of 29 amino acids and has a cyclic peptide backbone; (B) the primers used in the PCR reactions. Primers Kal1 (SEQ ID NO: 28) and Kal2 (SEQ ID NO: 29) correspond to amino acid residues 12 to 17 (SEQ ID NO: 42) or 29 to 5 (SEQ ID NO: 43) (see FIG. 1A). I represents inosine, Y represents C or T, and R represents A, C, T or G. The encoded amino acids are represented in single letter code and the introduced restriction enzyme sites are in italics; and (C) the PCR products. cDNA prepared from O. affinis leaf RNA was amplified with primers Kal1 and oligo dT-HindIII (lane 1) or primers Kal2 and oligo-dT-HindIII (lane 2) [SEQ ID NO: 30]. The amplified fragments were separated on a 2% w/v agarose gel and stained with ethidium bromide. Five major fragments were obtained, two from primer Kal1 (1-2) and three from primer Kal2 (3-5). Fragments 1 and 5 were subcloned and sequenced.
FIG. 2 is a presentation of the sequence of the 412 bp DNA fragment amplified from O. affinis cDNA with primers complementary to the Kalata B1 sequence (FIG. 1C, fragment 5). The 412 bp fragment has an open reading frame (SEQ ID NO: 7) that encodes the entire 29 amino acids of Kalata B1 [SEQ ID NO: 41] together with an additional four amino acids at the C-terminus (SEQ ID NO: 40). The 3′ untranslated region is shown in SEQ ID NO: 9. The primer sequences are shown in italics, stop codons are indicated by “*” and a region corresponding to the sequence of the mature peptide is underlined.
FIG. 3 is a photographic representation showing gel bot analysis of RNA from O. affinis leaves. (A) The RNA blot. The Kalata B1 cDNA (see FIG. 2) hybridized to a single RNA transcript of  ̃750 bases. (B) Identical gel to (A) stained with ethidium bromide to reveal the rRNA bands. Size markers were the 0.28-6.58 kb RNA markers from Promega.
FIG. 4 is a representation of nucleotide sequence (SEQ ID NO: 4) and predicted amino-acid sequence (SEQ ID NO: 5) of Oak1, the cDNA encoding Kalata B1 from O. affinis. The 5′ and 3′ ends of the DNA are shown in SEQ ID NO: 1 and SEQ ID NO: 6, respectively. The nucleotide sequence encoding the signal peptide and the corresponding amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 3, respectively. Only one strand with the polarity of the mRNA is shown. Nucleotides are numbered above the sequence. The amino acid sequence, shown in single letter code is numbered beginning with 1 for the predicted first amino acid in the precursor protein. The putative signal peptide is indicated by negative numbers. An amino acid sequence subjected to processing to give Kalata B1 is shaded. Arrows indicate potential processing sites. The underlined region at the N-terminus of the Kalata B1 domain (NT-conserved) is highly conserved in other Oak clones (see FIGS. 8A and 8B).
FIG. 5 is a representation of the nucleotide sequence (13) and predicted amino-acid sequence (SEQ ID NO: 14) of Oak2, the cDNA encoding Kalata B3 and B6 from O. affinis. Only one strand with the polarity of the mRNA is shown. Nucleotides are numbered above the sequence. The amino acid sequence, shown in single letter code, is numbered beginning with 1 for the predicted first amino acid in the precursor protein. The putative signal peptide is indicated by negative numbers (SEQ ID NO: 12) and is encoded by the nucleotide sequence set forth in SEQ ID NO: 11. An amino acid sequence subjected to processing to give Kalata B3 and B6 is shaded. Dark and light shading respectively highlights the sequence of Kalata B3 and B6. Arrows indicate potential processing sites. The underlined region at the N-terminus of the Kalata B3 and B6 domains (NT-conserved) is highly conserved in other Oak clones (see FIGS. 8 A and 8B). The untranslated 5′ and 3′ ends of the DNA is shown in SEQ ID NO: 10 and SEQ ID NO: 15, respectively.
FIG. 6 is a representation of the nucleotide sequence (SEQ ID NO: 19) and predicted amino-acid sequence (SEQ ID NO: 20) of Oak3, the cDNA encoding Kalata B7 from O. affinis. Only one strand with the polarity of the mRNA is shown. Nucleotides are numbered above the sequence. The amino acid sequence, shown in single letter code, is numbered beginning with 1 for the first predicted amino acid in the precursor protein. The putative signal peptide (SEQ ID NO: 18) is indicated by negative numbers and is encoded by the nucleotide sequence set forth in SEQ ID NO: 17. An amino acid sequence subjected to processing to give Kalata B7 is shaded. Arrows indicate potential processing sites. The underlined region at the N-terminus of the Kalata B7 domain (NT-conserved) is highly conserved in other Oak clones (see FIGS. 8 A and 8B). The untranslated 5′ and 3′ ends of the DNA is shown in SEQ ID NO: 16 and SEQ ID NO: 21, respectively.
FIG. 7 is a representation of the nucleotide sequence (SEQ ID NO: 25) and predicted amino-acid sequence (SEQ ID NO: 26) of Oak4, the cDNA encoding Kalata B2 from O. affinis. Only one strand with the polarity of the mRNA is shown. Nucleotides are numbered above the sequence. The amino acid sequence, shown in single letter code, is numbered beginning with 1 for the first predicted amino acid in the precursor protein. The putative signal peptide (SEQ ID NO: 24) is indicated by negative numbers and is encoded by SEQ ID NO: 23. An amino acid sequence subjected to processing to give Kalata B2 (shaded) is repeated three times. Arrows indicate potential processing sites. The underlined region at the N-terminus of the Kalata B2 domain (NT-conserved) is highly conserved in other Oak clones (see FIGS. 8 A and 8B). The untranslated 5′ and 3′ ends of the DNA is shown in SEQ ID NO: 22 and SEQ ID NO: 27, respectively.
FIG. 8 is a schematic diagram of (A) of the precursor proteins predicted from the Oak 1, 2, 3 and 4 clones showing the signal peptide, the regions corresponding to the mature kalata peptides (shaded), the region of 17 conserved amino acids on the N-terminal side of the kalata peptide sequence (N-T conserved, hatched), and (B) the sequence around the potential processing sites (SEQ ID NOs: 34-38). The mature cyclic peptide retains one copy of the Gly-Leu-Pro sequence, which may be derived entirely from one of the two flanking elements (shaded), or partially from both depending on the initial cleavage sites.
FIG. 9 is a diagrammatic representation showing the structure of Kalata B2 and a comparison with Kalata B1. (A) shows the circular backbone and cross-linking disulfide bonds of Kalata B2. The Cys residues making up these disulfide bonds are labelled I-VI. The arrows represent regions of beta strands. The side chains of two aromatic residues which are located on proximate turns are highlighted. (B) shows a superimposition of the backbone residues of Kalata B2 and Kalata B1 and demonstrates the similarity of their three dimensional structures.
FIG. 10 is a photographic representation of gel blot analysis of genomic DNA from O. affinis. Gel blot analysis of genomic DNA digested with HindIII, BamH1, Nde I and EcoRV and probed with radiolabelled Oak1 cDNA (FIG. 4). All enzymes gave at least twelve hybridizing bands. The Oak clones appear to belong to a multigene family with up to twelve members.
FIG. 11 is a representation showing bacterial expression of the precursor protein encoded by the Oak1 clone. (A) Total cell lysates were prepared at various time points (0-5 hr) post-induction with IPTG by removing 100 μl of cell culture which was lysed in SDS-sample buffer. The proteins were separated on a 12% w/v SDS polyacrylamide gel and stained with silver. A band of approximately the expected size (arrowed) appeared after IPTG induction. Lane numbers indicate hours after induction and broad range kaleidoscope markers (BioRad) were used. IMAC is induced protein purified by immobilized metal affinity chromatography (IMAC). Full-length Oak1 was expressed from the pQE.30 vector in an E. coli M15 cell line. B. RP-HPLC of the material that bound to the metal affinity column. The protein in the peak had the same mass as the protein predicted by the Oak1 cDNA together with the hexahistidine tag and henceforth will be called Kalata B1 precursor.
FIG. 12 is a representation showing immunoblot analysis with the antibody raised to the Kalata B1 precursor. (A) Purified kalata B1 (1 μg), Kalata B1 precursor (1 μg) and buffer soluble proteins from O. affinis leaves (100 μg) fractionated on a precast 4-12% bis-tris protein gel (Novex, San Diego, Calif., USA) and stained with silver. Markers are the SeeBlue (trademark) Pre-Stained standard from Novex. (B) Proteins in an identical gel after transfer to nitrocellulose (0.2μ) and immunoblotting with antiserum (1:1000) raised against the bacterially expressed Kalata B1 precursor (see FIG. 11). Antibody raised to the Kalata B1 precursor recognizes both the precursor and cyclic Kalata B1.
FIG. 13 is a graphical representation showing the effect of Kalata B1 on growth and development of H. punctigera larvae. (A) Survival of larvae fed on Haricot bean artificial diet containing Kalata B1 and the control diet. (B) Average mean weight of larvae fed on Kalata B1 and control diet. (C) Size of larvae after 16 days on artificial diet containing Kalata B1 ( ̃5 mm) or control diet ( ̃30 mm).
FIG. 14 illustrates the effect of cyclic and linear Kalata B1 on growth of H. armigera larvae. (A) Graphical representation of the growth of larvae fed on a cotton leaf artificial diet containing Kalata B1 at 0.15% w/v of diet (high cyclic), Kalata B1 at 0.03% w/v (low, cyclic), linear Kalata B1 at 0.15% w/v or no added Kalata B1 (control). (B) Relative size of larvae after 12 days. 1. Cyclic Kalata B1 (0.15% w/v), 2. Cyclic Kalata B1 (0.03% w/v), 3. Linear Kalata B1 (0.15% w/v), 4. Control.
FIG. 15 is a representation of the sequence of the 311 bp fragment amplified from Viola odorata cDNA with the primers Kal2 and oligo dT-HindIII (see FIG. 1). The 311 bp fragment has an open reading frame (SEQ ID NO: 31) that encodes 26 amino acids (SEQ ID NO: 32) of Kalata S (underlined) a cyclotide isolated from V. odorata (12). An additional four amino acids are located at the C-terminus of the Kalata S sequence. The primer sequences are shown in italics, the stop codon is indicated by “*” and the coding region is underlined. The untranslated 3′ end of the DNA is shown in SEQ ID NO: 34.
FIG. 16 is a photographic representation showing gel blot analysis of RNA from roots, leaves and stems of O. affinis. (A) The RNA blot. The Oak1cDNA (see FIG. 4) hybridized to a broad band of about 750 bases. (B) Identical gel to (A) stained with ethidium bromide to reveal the rRNA bands. Size markers were the 1 Kb Plus DNA Ladder (trademark) from Gibco BRL.



Table 1 is a summary of single and three letter abbreviations used throughout the specification are defined in Table 1.
Table 2 is a summary of amino acid and nucleotide sequence identifiers.







TABLE 1







Single and three letter amino acid abbreviations












Three-letter
One-letter



Amino Acid
Abbreviation
Symbol






Alanine
Ala
A



Arginine
Arg
R



Asparagine
Asn
N



Aspartic acid
Asp
D



Cysteine
Cys
C



Glutamine
Gln
Q



Glutamic acid
Glu
E



Glycine
Gly
G



Histidine
His
H



Isoleucine
Ile
I



Leucine
Leu
L



Lysine
Lys
K



Methionine
Met
M



Phenylalanine
Phe
F



Proline
Pro
P



Serine
Ser
S



Threonine
The
T



Tryptophan
Trp
W



Tyrosine
Tyr
Y



Valine
Val
V



Any residue
Xaa
X

















TABLE 2





SEQUENCE



IDENTIFIER
DESCRIPTION







SEQ ID NO: 1
Untranslated 5′ end of DNA encoding Kalata B1


SEQ ID NO: 2
Nucleotide sequence encoding signal peptide of Kalata



B1


SEQ ID NO: 3
Amino acid sequence of signal peptide of Kalata B1


SEQ ID NO: 4
Nucleotide sequence of cDNA encoding Kalata B1


SEQ ID NO: 5
Amino acid sequence of Kalata B1 encoded by



SEQ ID NO: 4


SEQ ID NO: 6
Untranslated 3′ end of DNA encoding Kalata B1


SEQ ID NO: 7
Partial nucleotide sequence of a 400 bp DNA fragment



amplified from O. affins cDNA


SEQ ID NO: 8
Partial amino acid sequence encoded by



SEQ ID NO: 7


SEQ ID NO: 9
Untranslated 3′ end of 400 bp DNA fragment from



O. affins cDNA


SEQ ID NO: 10
Untranslated 5′ end of DNA encoding Kalata B3 and



B6


SEQ ID NO: 11
Nucleotide sequence encoding signal peptide of



Kalata B3 and B6


SEQ ID NO: 12
Amino acid sequence of signal peptide of Kalata B3



and B6


SEQ ID NO: 13
Nucleotide sequence of cDNA encoding Kalata B3 and



B6


SEQ ID NO: 14
Amino acid sequence of Kalata B3 and B6


SEQ ID NO: 15
Untranslated 3′ end of DNA encoding Kalata B3 and



B6


SEQ ID NO: 16
Untranslated 5′ end of DNA encoding Kalata B7


SEQ ID NO: 17
Nucleotide sequence encoding signal peptide of



Kalata B7


SEQ ID NO: 18
Amino acid sequence of signal peptide of Kalata B7


SEQ ID NO: 19
Nucleotide sequence of cDNA encoding Kalata B7


SEQ ID NO: 20
Amino acid sequence of Kalata B7


SEQ ID NO: 21
Untranslated 3′ end of DNA encoding Kalata B7


SEQ ID NO: 22
Untranslated 5′ end of DNA encoding Kalata B2


SEQ ID NO: 23
Nucleotide sequence encoding signal peptide of



Kalata B2


SEQ ID NO: 24
Amino acid sequence of signal peptide of Kalata B2


SEQ ID NO: 25
Nucleotide sequence of cDNA encoding Kalata B2


SEQ ID NO: 26
Amino acid sequence of Kalata B2


SEQ ID NO: 27
Untranslated 3′ end of DNA encoding Kalata B2


SEQ ID NO: 28
Nucleotide sequence of Kal1 primer


SEQ ID NO: 29
Nucleotide sequence of Kal2 primer


SEQ ID NO: 30
Oligo-dT-HindIII nucleotide sequence


SEQ ID NO: 31
Nucleotide sequence of coding region of 311 bp



fragment amplified by Viola odorata cDNA


SEQ ID NO: 32
Amino acid sequence encoded by



SEQ ID NO: 31


SEQ ID NO: 33
Untranslated 3′ end of 311 bp fragment amplified



from Viola odorata cDNA









DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is predicated in part on the elucidation of the genetic basis behind the production of macrocyclic peptides.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell to form a cyclic backbone wherein said cyclic backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three dimensional structure of said backbone.
The term “knotted” is not to be limited by any mathematical or geometrical definition of the term “knot”. The knots contemplated by the present invention are such due to their similarity to a mathematical knot and/or by virtue of the intertwined folding of the molecule which results.
Preferably, the stabilized folded structure contains a knotted topology. Accordingly, the present invention is directed, therefore, to an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell to form a cyclic knotted peptide, polypeptide or protein. The amino acid sequence may also be cyclizable in an in vitro system comprising, for example, cyclizing enzymes or the chemical means for cyclization.
An “isolated nucleic acid molecule” is a nucleic acid molecule which has undergone at least one purification step from a biological sample. Purification steps include inter alia precipitation, centrifugation, chromatography, electrophoresis and/or filtration. The nucleic acid molecule may be single or double stranded RNA or DNA or an RNA:DNA hybrid.
The nucleic acid molecule may comprise naturally occurring nucleotide bases or the bases may be synthetic or chemical analogues of bases or be chemically modified such as a C-5 propyne or phosphorothiolate modification.
An “amino acid sequence” generally means a sequence of two or more amino acid residues. In terms of the cyclotides of the present invention, generally the amino acid sequence comprises at least from about 10 to about 150 amino acid residues, preferably from about 15 to about 100 amino acid residues and even more preferably from about 15 to about 50 amino acid residues, when in cyclic form. The amino acid sequence referred to herein may be considered a peptide or polypeptide and these terms are used interchangedly in the subject specification. A “polypeptide’ may also be considered a “protein”.
However, the nucleic acid molecule of the present invention may first encode a precursor, peptide, polypeptide or protein, generally in linear form. Such a precursor may comprise from about 50 to about 1000 amino acid residues or from about 50 to about 500 amino acid residues or from about 50 to about 300 amino acid residues.
The precursor amino acid sequence is derivatized to a smaller amino acid sequence which is then cyclized. Alternatively, the cyclization process may include a derivatization step. The cyclization step may also occur in vitro using isolated enzyme systems or using chemical means.
Reference to a “derivative” of the amino acid sequence includes the derivatization of a precursor sequence to a cyclizable sequence.
The present invention extends to the nucleic acid molecule encoding the cyclotide or its linear precursor. Furthermore, the ‘cyclotide” may be linear in the sense that it has free amino acid and carboxy termini but still folds into a knot arrangement. Such a linear form is regarded as a structural homologue of the cyclotide.
Accordingly, another aspect of the present invention contemplates an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of forming a structural homologue of a cyclic peptide, polypeptide or protein within a cell or a membrane of a cell to form a backbone wherein said backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three dimensional structure of said backbone wherein said backbone comprises free amino and carboxyl termini.
Reference herein to a “cyclic backbone” preferably includes a molecule comprising a sequence of amino acid residues or homologues thereof without free amino acid and carboxy and amino termini.
The cyclic backbone encoded by the nucleic acid molecule of the present invention comprises sufficient disulfide bonds, or chemical equivalents thereof, to confer a stabilized folded structure on the three dimensional structure of the cyclic backbone.
Preferably, the stabilized folded structure comprises a knotted topology.
In a preferred embodiment, the cyclic backbone comprises a cystine knot. A cystine knot occurs when a disulfide bond passes through a closed cyclic loop formed by two other disulfide bonds and the amino acids in the backbone. Such a cystine knot is referred to herein as “cyclic cystine knot” or “CCK”. Reference herein, however, to a cyclic cystine knot or a CCK includes reference to structural equivalents thereof which provide similar constraints to the three dimensional structure of the cyclic backbone. For example, appropriate turns and loops in the cyclic backbone may also be achieved by engineering suitable covalent bonds or other forms of molecular associations. All such modifications to the cyclic backbone which retains the three-dimensional knotted topology conferred by the cyclic cystine knot are encompassed by the present invention including such modifications to the nucleic acid molecule which encodes a modified cyclotide. Furthermore, although a cyclic cystine knot is characterized by a knot formed on three disulfide bonds, the present invention extends to molecules comprising only two disulfide bonds. In such a case, the cyclic peptide, polypeptide or protein may need to be further stabilized using other means or the molecule may retain suitable activity despite a change in three-dimensional structure caused by the absence of a third disulfide bond. Reference herein to a “knotted topology” is not to be construed as limiting the invention to such a topology alone since the instant invention extends to any stabilizing folded structure. Furthermore, the cyclic backbone may comprise more than three disulfide bonds such as occurring in a double or multiple cystine knot arrangement or in a single cystine knot arrangement supplemented by one or two additional disulfide bonds.
Another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes an amino acid sequence capable of forming a cyclic backbone wherein the cyclic backbone comprises the structure:—




wherein

    
    
        C is cysteine;
        each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe] and [XV1 . . . XVf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and
        wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and each of a to f may be the same or different and range from 1 to about 20.
    
    


Preferably, each of a to f ranges from 1 to about 10.
In a particularly preferred embodiment, the present invention provides a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes an amino acid sequence capable of forming a cyclic backbone wherein the cyclic backbone comprises the structure:—




wherein

    
    
        C is cysteine;
        each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe] and [XV1 . . . XVf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and
        wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and wherein a is from about 3 to about 6, b is from about 3 to about 5, c is from about 2 to about 7, d is about 1 to about 3, e is about 3 to about 6 and f is from about 4 to about 9.
    
    


In an even more particularly preferred embodiment, the present invention provides a nucleic acid molecule an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell in an in vitro cyclizing system to form a cyclic backbone wherein the cyclic backbone comprises the structure:—




wherein

    
    
        C is cysteine;
        each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe] and [XV1 . . . XVf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and
        wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and each of a is about 3, b is about 4, c is about from 4 to about 7, d is about 1, e is about 4 or 5 and f is from about 4 to about 7.
    
    


In still an even more particularly preferred embodiment, the present invention provides a nucleic acid molecule an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell in an in vitro cyclizing system to form a cyclic backbone wherein the cyclic backbone comprises the structure:—




wherein

    
    
        C is cysteine;
        each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc], [XIII1 . . . XIIId], [XIV1 . . . XIVe] and [XV1 . . . XVf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and
        wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and wherein a is about 6, b is about 5, c is about 3, d is about 1, e is about 5 and f is about 8.
    
    


The invention extends to and includes peptide, polypeptide and protein sequences which have not been acted upon by an enzyme system separate from the molecule itself. For example, the present invention extends to autocatalytic cyclization.
The cyclization of the amino acid sequence may occur in a cell such as a plant cell or cell membrane which naturally contains the cyclization enzyme or enzymes. An in vitro system may also be employed with isolated enzyme(s) capable of cyclizing target molecules. Chemical means may also be employed to facilitate cyclization. Alternatively, cells may be engineered to express the genes encoding the cyclization enzyme or enzymes. In relation to the former, preferred cells are whole plants, callus or cell lines or membranous preparations of cells from the Rubiaceae, Violaceae or Cucurbitaceae plant families. However, any other plant or part of a plant which contains the requisite cyclization enzymes are encompassed by the present invention. In relation to engineered cells, these may be plant, animal, insect, fungal, yeast or microbial cells. The cyclization enzyme(s) may be encoded by genetic sequences resident on a plasmid or vector or the genetic sequences may be integrated into the chromosome of the cells. A genetic construct may also be introduced comprising nucleotide sequences encoding an amino acid sequence to the cyclized and the enzyme(s) required for cyclization.
The gene or genes required to encode the cyclization enzymes may be isolated by any number of means including differential display, yeast two hybrid systems, immunological screening techniques and a variety of visual display techniques. In one example, a nucleic acid molecule of the present invention is manipulated to include a nucleotide sequence which encodes a reporter molecule capable of giving an identifiable signal. The reporter gene sequence is inserted into or fused to a nucleotide sequence encoding a precursor form of the cyclotide. Examples of suitable reporter molecules include luciferase and β-galactosidase. The reporter molecule may also encode an amino acid sequence for which an antibody, labelled with a reporter molecule, may interact.
The modified nucleic acid molecule can then be transferred by, for example, transformation, electroporation, conjugation or Agrobacterium-mediated transfer to, for example, a bank of cells carrying a genomic library from a plant known to contain the cyclising enzyme or enzymes. When a modified nucleic acid molecule comprising a reporter gene sequence is introduced into a cell comprising the cyclizing enzymes, the precursor sequence is processed and the reporter molecule-encoding portion is cleaved off. As a result, the cells would not produce a detectable signal. Such cells would then be selected for further analysis. Agrobacterium-mediated transformation may be via embryonic or organogenic callus.
Many other approaches may be used to screen for and select clones encoding the cyclizing enzymes or to directly identify the enzyme(s). For example, the cyclizing enzyme(s) may be identified using fluoro or colormetric substrates designed based on knowledge of the precursor sequence described herein. In one embodiment, linear peptides are produced which comprise a cleaveable sequence with a colored reagent (e.g. PNA) that would get released and be detectable on treatment with plant-extract containing the enzyme(s). This provides a very powerful selection protocol for plant material containing cyclizing enzyme(s).
Accordingly, another aspect of the present invention contemplates a method of identifying nucleic acid molecules which encode one or more enzymes required for cyclization of an amino acid sequence said method comprising obtaining a nucleic acid molecule which encodes a precursor form of an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein; introducing or fusing to said nucleic acid molecule, a nucleotide sequence which encodes a reporter molecule capable of providing a detectable signal wherein said nucleotide sequence is inserted or fused to a portion of the nucleic acid molecule which is cleaved off prior to or during cyclization; introducing said nucleic acid molecule comprising the nucleotide sequence encoding the reporter molecule into a bank of cells carrying a DNA library comprising all or part of genomic DNA or cDNA from a plant which carries the enzyme or enzymes required for cyclization of an amino acid sequence; screening for and selecting cells which do not synthesize the reporter molecule.
In a preferred embodiment, the plant is from the Rubiaceae, Violaceae or Cucurbitaceae family.
The cyclizing enzyme(s) may also be useful in cyclizing smaller peptides and are useful, for example, in the generation of combinatorial chemistry libraries of small (e.g. 5-30 amino acids) cyclic peptides. These may have a range of applications such as pharmaceutical applications.
The present invention further contemplates a genetically modified cell or cells or a plant or animal comprising said genetically modified cells, said cells comprising a nucleic acid molecule having a nucleotide sequence or complementary nucleotide sequence which encodes an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein. The peptide, polypeptide or protein may be cyclized in vivo or in vitro.
The cells may also be in the form of cells or cell lines or cell cultures.
The present invention permits the manipulation of the nucleic acid molecules to introduce particular functional traits into the cyclized molecules or their linear forms or their precursor forms. A “trait” includes an activity, molecule interacting ability or some other attribute which has the capacity to alter a phenotype. For example, the peptides, polypeptides or proteins may be manipulated to introduce modulating activity of, for example, calcium channel-binding useful in the treatment of pain or a stroke, C5a binding activity useful as an anti-inflammatory agent, proteinase inhibitor activity in plants or animals, antibiotic activity, viral activity (e.g. of HIV or hepatitis virus), microbial activity, fungal activity, cytokine binding and blood clot inhibiting ability amongst other properties. Alternatively, the cyclic molecules themselves comprise useful traits such as being active against plant pests or pathogens. A plant pest or pathogen includes an insect, arachnid, microorganism, virus and a fungus. The term “pathogen” refers to any biological agent capable of interfering with biological function of said crop plants such that potential agronomic output of said crop plants is reduced.
Accordingly, the present invention contemplates a method of incorporating an amino acid sequence conferring a particular trait into a cyclic peptide, polypeptide or protein, said method comprising fusing or introducing a nucleotide sequence encoding said amino acid sequence to or into a second nucleotide sequence wherein said second nucleotide sequence encodes a peptide, polypeptide or protein which peptide, polypeptide or protein or a derivative therefor is capable of being cyclized into a knotted peptide, polypeptide or protein.
The present invention extends to an antibody that can be used to identify and detect said peptide which is cyclized or not. The invention further extends to the use of the antibody as a primary antibody to detect the said peptide sequence after gel electrophoresis and Western blot transfer.
The present invention comprises a peptide sequence that can be processed from a larger polypeptide sequence. More specifically, the excised polypeptide sequence is flanked by the amino acid sequence triplet GLP at the N-terminal end of the peptide to be cyclized and at the C-terminal end of the peptide to be cyclized. More specifically, the invention refers to a peptide sequence which can be cleaved at the peptide bond immediately prior to either glycine, leucine, proline or valine in either of the GLP sequences flanking the peptide to be excised (see FIG. 8B). All these processing sites are encompassed by the present invention. In particular, the present invention contemplates a range of processing sites which may occur naturally or be introduced. For example, different processing sites may be introduced such that depending on the organelle or tissue targeted, the peptide, polypeptide or protein may be differently processed.
The introduced amino acid sequence is referred to herein as a “heterologous” amino acid sequence.
Another aspect of the invention contemplates an isolated nucleic acid molecule comprising the following nucleotide sequence:—

    
    
        [X1 . . . Xb[n1n2 . . . na]y1 . . . yc]d 

wherein

        [n1n2 . . . na] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein; and
        X1 . . . Xb and y1 . . . yc represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d may be any number and when d is >1, the amino acid sequence may be unique for each integer of d.
    
    


The nucleic acid molecule according to this aspect of the present invention may be regarded as a hybrid nucleotide sequence comprising the structure:—

    
    
        j1 . . . je[X1 . . . Xa[n1n2 . . . na]y1 . . . yc]dq1 . . . qf 

wherein

        [n1n2 . . . na] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein;
        X1 . . . Xb and y1 . . . yc represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d may be any number and when d is >1, the amino acid sequence may be unique for each integer of d; and
        j1 . . . je and q1 . . . qf represent nucleotide sequences encoding a peptide, polypeptide or protein capable of directing the peptide, polypeptide or protein to a cellular compartment or organelle where a, b, c, d, e and f may be any number, where d is >1, the amino acid sequence may be unique for each integer of d.
    
    


In a related embodiment, there is provided an isolated nucleic acid molecule comprising the following nucleotide sequence:—

    
    
        [X1 . . . Xb[n1n2 . . . (k1 . . . kδ)λna]y1 . . . yc]d 

wherein

        [n1n2 . . . na] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein;
        X1 . . . Xb and y1 . . . yc represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d, δ and λ may be any number and when d or λ is >1, the amino acid sequence may be unique for each integer of d and λ;
        k1 . . . kδ represent a nucleotide sequence encoding an amino acid sequence conferring a particular activity or other trait.
    
    


In yet another related embodiment, the present invention is directed to an isolated nucleic acid molecule comprising the following nucleotide sequence:—

    
    
        [n1n2 . . . (n11n21 . . . nγ1) . . . na]m 

wherein

        [n1n2 . . . na] and (n11n21 . . . nγ1) represent polynucleotide sequences encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein; and
        γ and a and m may be any number and when m is >1, the amino acid sequence may be unique for each integer of m.
    
    


Further, the invention contemplates an isolated nucleic acid molecule comprising the following nucleotide sequence:—

    
    
        j1 . . . je[X1 . . . Xb[n1n2 . . . (n11n21 . . . (k1 . . . kδ)λna1)mna]y1 . . . yc]dq1 . . . qf 

wherein

        [n1n2 . . . na] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein;
        X1 . . . Xb and y1 . . . yc represents a polynucleotide sequence capable of encoding an amino acid sequence where a and b and c and d and e may be any number and when d is >1, the amino acid sequence may be unique for each integer of d;
        j1 . . . je and q1 . . . qf represents a nucleotide sequence encoding a peptide, polypeptide or protein capable of directing the peptide, polypeptide or protein to a cellular compartment or organelle;
        k1 . . . kδ represents a nucleotide sequence encoding an amino acid sequence conferring a particular activity or other trait;
        λ and m and d may be any number and when λ and m and d are each >1, the amino acid sequence may be unique for each integer of λ, m and d.
    
    


The “activity” may inter alia be a therapeutic activity, an enzymic activity or an activity useful as a laboratory reagent. Protease inhibitor activities, for example, may be used to generate plants (e.g. crops) resistant to pathogens. Alternatively, protease activities may be used to produce stable molecules useful in industrial grade washing compositions.
In a particularly preferred embodiment, the activity confers protection to a plant or animal cell from pathogen infestation. Examples of pathogens include insects, spiders, and other arachnids, microorganisms, viruses and fungi. Such an activity may also be exhibited by the cyclic peptide, polypeptide or protein without need for the introduction of an additional amino acid sequence. The present invention further extends to linear forms and precursor forms of the peptide, polypeptide or protein which may also have activity or other utilities. Various agricultural applications of the cyclic molecules or their linear forms are particularly contemplated by the present invention. For example, the present invention extends to engineering crop plants (such as cotton) to be resistant to pathogens (e.g. insects).
The present invention further provides genetic constructs comprising the nucleic acid molecules of the present invention. Such a genetic construct is particularly useful for expressing the nucleic acid molecule to produce linear or precursor forms of the cyclizable amino acid sequence. In this case, the genetic construct also comprises one or more promoters operably linked to the nucleotide sequences. Genetic constructs suitable for use in plants are particularly preferred. The genetic constructs may encode linear forms only of the peptides which are then subsequently circularized in vitro using, for example, enzyme(s) or chemical means. Alternatively, cell or cell membrane systems may be employed.
The genetic construct of the present invention may comprise a sequence of nucleotides or be complementary to a sequence of nucleotides which comprise one or more of the following: a promoter sequence, a 5′ non-coding region, a cis-regulatory region such as a functional binding site for a transcriptional regulatory protein or a translational regulatory protein, an upstream activator sequence, an enhancer element, a silencer element, a TATA box motif, a CCAAT box motif, an open reading frame, a transcriptional start site, a translational start site, and/or nucleotide sequence which encodes a leader sequence. The genetic construct of the present invention also encodes cyclizable peptide, polypeptide or protein. Furthermore, the genetic construct may comprise a cassette which may be used to insert a nucleotide sequence to be inserted into or fused to a cyclized peptide, polypeptide or protein. Furthermore, the nucleotide sequence to be inserted may consist of polynucleotide units called codons. A codon consists of three nucleotide bases wherein the sequence of the three nucleotide bases defines a specific amino acid. The invention extends to the use of any codon, triplet or polynucleotide sequence known to encode a specific amino acid. Furthermore, the invention extends to any polynucleotide sequence that defines a peptide sequence or polypeptide that can be fused to the inserted sequence for the purpose of targeting, transporting or regulating the expression of the polypeptide sequence to which it is fused. In one particular embodiment, a vacuole or other cellular organelle is targeted.
The term “5′ non-coding region” is used herein in its broadest context to include all nucleotide sequences which are derived from an upstream region of an expressible gene, other than those sequences which encode amino acid residues which comprise the polypeptide product of the gene, wherein the 5′ non-coding region confers or activates or otherwise facilitates, at least in part, expression of the gene.
The term “gene” is used in its broadest context to include both a genomic DNA region corresponding to the gene as well as a cDNA sequence corresponding to exons or a recombinant molecule engineered to encode a functional form of a product. A gene includes any sequence of nucleotides which may be transcribed into a mRNA molecule. Use of the term “gene” is not to place any structural or functional constraints on the scope of the present invention.
As used herein, the term “cis-acting sequence” or “cis-regulatory region” or similar term shall be taken to mean any sequence of nucleotides which is derived from an expressible genetic sequence wherein the expression of the first genetic sequence is regulated, at least in part, by said sequence of nucleotides. Those skilled in the art will be aware that a cis-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of any structural gene sequence.
Reference herein to a “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is usually, but not necessarily, positioned upstream or 5′, of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
In the present context, the term “promoter” is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a structural gene or other nucleic acid molecule, in a cell. Preferred promoters according to the invention may contain additional copies of one or more specific regulatory elements to further enhance expression in a cell and/or to alter the timing of expression of a structural gene to which it is operably connected.
The term “operably connected” or “operably linked” in the present context means placing a structural gene under the regulatory control of a promoter which then controls expression of the gene. Promoters and the like are generally but not necessarily positioned 5′ (upstream) to the genes which they control. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the genetic sequence or promoter at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting, i.e., the gene from which the genetic sequence or promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived.
The genetic construct(s) of the present invention may be introduced into a cell by various techniques known to those skilled in the art. The technique used may vary depending on the known successful techniques for that particular cell.
Techniques for introducing recombinant DNA into cells such as plant cells include, but are not limited to, transformation using CaCl2 and variations thereof, direct DNA uptake into protoplasts, PEG-mediated uptake to protoplasts, electroporation, microinjection of DNA, microparticle bombardment of tissues or cells, vacuum-infiltration of tissue with nucleic acid, and T-DNA-mediated transfer from Agrobacterium to the plant tissue.
For microparticle bombardment of cells, a microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf (U.S. Pat. No. 4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.
Examples of microparticles suitable for use in such systems include 0.1 to 10 μm and more particularly 1 to 5 μm tungsten or gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
In a particularly preferred embodiment, the nucleotide sequence encoding the cyclizable amino acid sequence or its precursor comprises the amino acid sequence substantially as set forth in <400>2, <400>4 or <400>6 or an amino acid sequence having at least 60% similarity thereto.
Preferably, the nucleotide sequence is substantially as set forth in <400>1, <400>3 or <400>5 or a sequence having at least 60% similarity thereto or capable of hybridizing thereto under low stringency conditions at 42° C.
The term “similarity” as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al. (7). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (8).
The terms “sequence similarity” and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity”, for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, “sequence identity” will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30° C. to about 42° C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out Tm=69.3+0.41 (G+C) % (9). However, the Tm of a duplex DNA decreases by 1° C. with every increase of 1% in the number of mismatch base pairs (10). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6×SSC buffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.; high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.
The present invention further contemplates a genetically modified plant which comprises a nucleotide sequence which encodes an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein and which confers on said plant a trait not present in the same species or variety of plant prior to genetic modification. The plant may also comprise one or more nucleotide sequences which encode one or more cyclizing enzymes.
The genetically modified plants of this aspect of the present invention include plants which are resistant to certain pathogens. Crop plants resistant to pathogens are particularly preferred. Crop plants include but are not limited to cotton, cereal crops, vegetable crops, seed crops and flowering crops.
Yet another aspect of the present invention provides the use of a nucleic acid molecule encoding an amino acid sequence, which amino acid sequence or a derivative or precursor form thereof is capable of being cyclized into a knotted peptide, polypeptide or protein, in the manufacture of a transgenic or genetically modified plant capable of producing said cyclic knotted peptide, polypeptide or protein.
The present invention is further described by the following non-limiting Examples.
Example 1
Plant Material
Oldenlandia affinis DC and Viola odorata were grown under standard glasshouse conditions.
Example 2
RNA Isolation
RNA was prepared from various tissues of O. affinis using TRIzol (trademark) reagent and the protocol from Gibco BRL (see Gibco BRL form #3796, TRIzol (trademark) Reagent Total RNA Isolation Reagent).
Example 3
Methods for Isolating a Partial cDNA Clone Encoding Kalata B1
Single stranded cDNA was prepared from O. affinis leaf RNA using the Gibco BRL RT-PCR kit and an oligo-dT primer according to the manufacturer's instructions. The cDNA produced was amplified by the polymerase chain reaction using one of two degenerate primers and oligo-dT (Bresatec). The two degenerate primers and the encoded protein sequence are shown in FIG. 1B. The oligonucleotides were dissolved in milliQ water to a final concentration of 200 μM. The PCR reaction was performed using a profile of 30 cycles of 94° C. (3 min), 37° C. (3 min) and 70° C. (3 min). After 30 cycles, there was a final extension step at 72° C. for 10 min. PCR products were separated on 2% w/v agarose gels in TBE (45 mM Tris Borate, 1 mM EDTA). The QIAGEN Gel extraction kit was used to purify the amplified fragments which were subsequently cloned into the pBluescript SK+ vector (Stratagene) and sequenced by SUPAMAC (Sydney University and Prince Albert Macromolecular Analysis Centre, Sydney, Australia), using T3 and T7 primers.
Example 4
Preparation and Screening of the O. affinis cDNA Library
Total RNA (1 mg) was prepared from leaves and stem and mRNA was separated using the PolyATract (trademark) I (Promega) mRNA isolation system. Five microgram of mRNA was used to produce the cDNA library in a Lambda-Zap vector using the Stratagene ZAP-cDNA and Gigapack cloning kits according to manufacturer's instructions. The amplified library was screened using a 32P-labelled DNA fragment corresponding to bases 7-105 of the partial Kalata B1 clone (FIG. 2) as probe. After a second round of screening, hybridizing clones were chosen for sequence analysis. Excised phagemids were transformed into XL1-Blue (Stratagene) E. coli cells via electroporation using a GenePulsar electroporation apparatus (BioRad). Plasmid was isolated using the alkaline lysis protocol (11). Sequence analysis was performed by SUPAMAC (Sydney University and Prince Albert Macromolecular Analysis Centre, Sydney, Australia), using T3 and T7 primers. Analysis of DNA sequences was performed using SeqEd (Applied Biosystems). The Oak1 and Oak 2 clones were isolated as described above. The Oak 3 and Oak 4 clones were isolated using an identical procedure except the full length Oak1 cDNA was used as probe.
Example 5
RNA Blots
Total RNA (10 μg) was fractionated on 1.2% w/v agarose gels in the presence of formaldehyde and transferred to HyBond N+ (Amersham) as described by Sambrook et al. (11). Prehybridization (at 42° C.) and hybridization (16 hr at 42° C.) was performed in 5×SSPE (0.9 M NaCl, 50 mM NaH2PO4.2H2O, 5 mM EDTA), 1% w/v SDS, 5×Denhardt's solution [0.1% w/v Ficoll, 0.1% w/v BSA fraction V, Sigma], 0.1% w/v polyvinylpyrrolidone, 50% w/v deionised formamide and 100 μg/ml Herring sperm DNA. The membrane was probed with either the Kalata B1 clone (FIG. 2) or the Oak1 cDNA0 (FIG. 4), unbound probe was removed by washing three times with 2×SSC and 1% w/v SDS at 42° C. for 10 min. Hybridizing RNA was visualized after exposure to a phosphoimager screen for 15 hr using a model 400B Phosphorimager (Molecular Dynamics) and ImageQuant (trademark) software.
Example 6
DNA Blots
Genomic DNA was isolated from fresh leaf material (1.5 g) as described in the QIAGEN Genomic Tip protocol. Genomic DNA (10 μg) was digested with restriction enzymes; HindIII, BamH1, Nde1 and EcoRV (Promega, 5 units) and separated on a 0.7% agarose gel in the presence of ethidium bromide and TBE buffer (11). The DNA was transferred to an N+ nitrocellulose membrane as described (11) and the blot was probed with the full-length Oak1 cDNA. The blot was prehybridized, hybridized and washed as described for the RNA blots. Hybridizing DNA was visualized using a model 400B Phosphorimager (Molecular Dynamics) and ImageQuant (trademark) software.
Example 7
Bacterial Expression of the Protein Encoded by Oak1
The cDNA encoding the Kalata B1 precursor protein was PCR amplified from the Oak1cDNA using oligonucleotide primers complementary to bases 61-75 (forward primer) and 361-372 (reverse primer). The amplified fragment was cloned into the pGEM-T-Easy vector (Promega) before it was excised and subcloned into pQE-30 (QIAGEN) to create pQKB1. Eschericia coli m15 cells containing the pREP-4 plasmid were transformed with pQKB1 and grown in Luria broth containing ampicillin (100 μg/ml) and kanamycin (12.5 μg/ml) before induction with IPTG (1 mM). Cells were then pelleted by centrifugation and resuspended in sample buffer (12) and heated before analysis by SDS-PAGE. Alternatively, the cells were suspended in a lysis buffer (50 mM Tris-HCl pH 8.5, 2 mM EDTA, 50 μg/ml lysozyme and 10% v/v Triton X-100; 5 ml of lysis buffer/g of cells) before incubation at 37° C. for 15 min. Cell lysate was then mixed with MgCl2 (10 mM final concentration) and DNase 1 (Roche, 10 μg/ml final concentration) and insoluble material was collected by centrifugation (13,000 rpm, 15 min, 4° C.). The insoluble protein pellet was washed several times with 0.5% Triton X-100, 10 mM EDTA before a final wash in distilled water. The proteins in the pellet were dissolved in denaturing lysis buffer (10 mM Tris-HCl pH 8.5, 100 mM NaCl, 8 M urea) before chromatography on an immobilized metal affinity column (TALON (registered trademark) metal affinity resin, Clontech, Palo Alto, Calif., USA) according to the procedure described by the manufacturer for Batch/Gravity-Flow Column Purification (Clontech user manual Protocol #PT1320-1, version #PR96975). Protein eluted from the column was analyzed by SDS-PAGE, RP-HPLC (reversed phase high performance liquid chromatography) or ESMS (electrospray ionization mass spectrometry).
Example 8
Reverse Phase High Pressure Liquid Chromatography (RP-HPLC) and Mass Spectrometry
RP-HPLC was performed on a Brownlee RP300 C8 analytical column (4.6×100 mm) using a Waters model 510 pump and a Waters model 481 UV detector. Samples were applied in 0.1% v/v trifluoroacetic acid (Buffer A) and were eluted with 60% v/v acetonitrile, 0.089% v/v trifluoroacetic acid (Buffer B) according to a gradient of 0-100% Buffer B over 30 min with a flow rate of 1 ml/min. Eluted protein was detected by absorbance at 215 nm. The molecular mass of the RP-HPLC purified protein was determined by electrospray ionization mass spectrometry (ESMS) using a Perkin-Elmer Sciex API-300 triple quadrupole mass spectrometer fitted with a micro-ionspray ion source.
Example 9
Bioassays with Artificial Diets
Helicoverpa punctigera larvae were raised on artificial diets based on Haricot beans (Teakle et al. (13)). One litre of diet was composed of 234 g Haricot beans, 14 g agar, 700 ml water, 35 g Tortula yeast, 50 g wheatgerm, 3.5 g ascorbic acid, 1.1 g sorbic acid, 2.2 g p-hydroxybenzoic acid methyl ester, 0.2 g ampicillin, 0.2 g streptomycin, 16 mg prochloraz. The beans were soaked overnight in water, drained and homogenized to a fine paste. Wheatgerm, yeast and 300 ml of water were added. The agar was dissolved in 400 ml of boiling water and added to the mixture. The mixture was cooled to 50° C. before the addition of the remaining ingredients. The blended diet was poured into trays and after setting was used immediately or stored at −20° C. for no longer than two weeks. The test diet was supplemented with the Kalata B1 peptide (0.825 μmol/g of diet). Twenty newly emerged neonates were added to each diet and mortality was recorded every two days. Weight gain was recorded at the sixth day and then every second day thereafter. The larvae were reared in 1.5 ml eppendorfs microfuge tubes (one larva/tube) until day eight when they were transferred to individual plastic containers with lids (Solo (trademark) plastic portion cups, 28 ml) at the eight day. Larvae were fed small amounts of diet (40 mg) initially that was replaced as required to provide a continuous supply. The larvae were kept in a temperature controlled room at 25±1° C., 16:8 (L:D).
Helicoverpa armigera larvae were raised on an artificial diet based on cotton leaves. One hundred ml of cotton leaf artificial diet was composed of 3 g cotton leaf powder (see below), 2 g Tortula yeast, 2.4 g wheat germ, 3.2 g ascorbic acid, 0.08 g sorbic acid, 0.16 g paraben (mould inhibitor), 0.08 ml linseed oil, 0.16 ml wheatgerm oil, 0.028 g ampicillin, 0.028 g streptomycin, 3.2 g agar and 80 ml water. Cotton leaf powder was prepared from freshly picked young, healthy cotton leaves which had been rapidly frozen in liquid nitrogen and freeze dried before they were ground to a fine powder using a mortar and pestle. Test diets contained the cyclic Kalata B1 peptide at 0.15% w/v of diet (high kalata) or 0.03% w/v of diet (low kalata), linear peptide (backbone chain opened between residues 7 and 8 [see FIG. 1A] at 0.15% w/v of diet. The control diet contained casein at 0.15% w/v of diet. The diet was mixed and dispensed as described for the Haricot bean diet and the bioassay was conducted in the same manner.
Example 10
Isolation of a Partial cDNA Encoding Kalata B1
As Kalata B1 is a cyclic protein of only 29 amino acids and the N-terminus was unknown, two degenerate primers were designed to amplify part of the encoding DNA (see FIG. 1). Five PCR amplified products were obtained using these primers in combination with the oligo-dT-HindIII primer (FIG. 1C). The 412 bp fragment produced from primer Kal2 (fragment 5, FIG. 1C) has a 3′ untranslated region of 267 bp and a poly A tail of 32 bp together with the complete coding sequence of Kalata B1 (FIG. 2). When used as a probe on RNA blots containing O. affinis leaf RNA, the partial Kalata B1 clone hybridised to an RNA transcript of about 750 bases, suggesting the cyclic peptide is derived from a larger precursor protein (FIG. 3).
Example 11
Isolation of a Full Length cDNA Clone for Kalata B1 and a Second cDNA Clone Encoding Two Kalata Related Peptides
The cDNA library prepared from leaf and stem mRNA was screened using the partial Kalata B1 cDNA as probe. Two full length clones were obtained, the first designated OaK1 (for O. affinis Kalata B1) was 725 bp long and encodes a predicted protein of 124 amino acids (FIG. 4). The 29 amino acid Kalata B1 sequence is embedded in a precursor protein which has a typical endoplasmic reticulum (ER) signal sequence of 20 amino acids. It is likely that the precursor enters the secretory pathway where folding and disulphide bond formation occurs prior to the cyclization and cleavage events that release the mature cyclic peptide. The B1 sequence is preceded by about 70 amino acids at the N-terminus and four to seven amino acids at the C-terminus. All six cysteines in the precursor are located in the B1 sequence. The predicted precursor has no potential N-glycosylation sites and, hence, has an expected mass of 11.18 kDa.
The second cDNA clone, designated Oak2 has an insert of 843 bp and unlike the first clone is predicted to encode two Kalata B1 related sequences which we have called B3 and B6 (FIG. 5). The predicated protein also has typical ER signal sequence of 20 amino acid which is followed by a 46 to 49 amino acid sequence before the first Kalata sequence (B6) is encountered. This peptide is separated from the Kalata B3 sequence by about 25 amino acids. The B3 sequence is flanked four amino acids at the C-terminus (SAAA SEQ ID NO:39) which are similar to those which flank B1 (SLAA SEQ ID NO: 40) in the protein encoded by the Oak1 clone. Like the precursor encoded by the Oak1 clone, the precursor encoded by the Oak2 clone has no potential N-glycosylation sites and all cysteine residues are confined to the Kalata peptide sequences. After removal of the potential ER signal sequence the precursor encoded by the Oak1 clone has a predicted mass of 14.56 kDa. The size of both clones is consistent with the size of the hybridizing transcript detected in the Northern analysis of leaf RNA (FIG. 3).
The third clone designated Oak3 was 677 bp long (FIG. 6) and encodes a predicted protein of 111 amino acids. It has only one Kalata B1 related sequence that has been called B7.
The fourth clone designated Oak4 has an insert of 993 bp and encodes a predicted protein of 210 amino acids (FIG. 7). This protein has three identical sequences that are related to Kalata B1. This sequence has been called Kalata B2.
A schematic diagram of the precursor proteins predicted from the Oak 1, 2, 3 and 4 clones is given in FIG. 8A. Cyclic peptides with the same sequence as Kalata B1, B2 and B3 have been isolated from the leaves of the O. affinis plant (14) (Example 12). The inventors conclude that these peptides are derived by proteolytic cleavage of a precursor protein and formation of a new peptide bond. That is, it is likely that B1, B2 and B3 cyclotides are produced from precursor proteins encoded by the Oak1, Oak4 and Oak2 clones respectively.
Each of the B1, B3, B6 and B7 peptides in the predicted precursor proteins (FIG. 8B) is flanked on both sides by the highly conserved sequence-1-Gly-2-Leu-3-Pro-4-. The circularization process thus involves specific ligation at the same cleavage site within both flanking sequences (one of the four peptide bonds shown) and ligation of the new N- and C-termini. The mature cyclic peptide retains one copy of the Gly-Leu-Pro sequence, which may be derived entirely from one of the original flanking elements, or partially from both depending on the initial cleavage sites (FIG. 8A).
The protein encoded by the Oak4 clone offers further insight into the potential processing site. Unlike the proteins encoded by the other three clones, the Oak4 protein has three copies of a Kalata like sequence. This sequence (B2) is flanked by Gly-Leu-Pro at the N-terminus and Ser-Leu-Pro at the C-terminus (FIG. 8B). The B2 peptide isolated from the plant (14) (Example 12) has retained the Gly-Leu-Pro sequence. Processing thus appears to have occurred at the peptide bonds preceding the glycine and the serine (see FIG. 8B).
Example 12
Isolation and Structure Determination of Kalata B2
Kalata B2 was isolated from aerial parts of O affinis by extraction with dichloromethane/methanol (50:50 v/v) and purified using reverse phase HPLC (Vydac C18 column). Gradients of CH3CN in H2O (0.1% trifluoroacetic acid, v/v) were employed in the purification. The purified Kalata B2 was reduced with an excess of tris-carboxyethyl phosphine, TCEP, and alkylated with maleimide. The reduced and alkylated peptide was cleaved with Endo-Glu C in ammonium acetate buffer at pH 7.7 for 2 hours and then purified by reverse phase HPLC. The cleaved peptide was N-terminally sequenced using Edman degradation on an Applied Biosystems 477A Protein Sequencer.
The structure of Kalata B2 was determined using NMR spectroscopy and simulated annealing calculations. Samples for 1H NMR measurement contained  ̃1.5 mM peptide in 90% H2O/10% D2O (v/v) at pH 3.6. Spectra were recorded at 290K, 298K and 305K on a Bruker ARX-500 spectrometer equipped with a shielded gradient unit and on a Bruker DRX-750 spectrometer. The following homonuclear 2D NMR spectra were recorded in phase-sensitive mode using time-proportional phase incrementation for quadrature detection in the t1: TOCSY using a MLEV-17 spin lock sequence with an isotropic mixing period of 80 ms; NOESY with mixing times of 200 ms, 250 ms and 300 ms; double quantum filtered DQF-COSY and E-COSY. For DQF-COSY and E-COSY spectra solvent suppression was achieved using selective low-power irradiation of the water resonance during a relaxation delay of 1.8 ms. Water suppression for NOESY and TOCSY experiments was achieved using a modified WATERGATE pulse sequence. Spectra were acquired over 6024 Hz with 4096 complex data points in F2 and 512 increments in the F1 dimension, with 16 to 64 scans per increment. Spectra were processed on a Silicon Graphics Indigo workstation using. UXNMR (Bruker) software. The t1 dimension was zero-filled to 2048 real data points and 90° phase-shifted sine bell window functions were applied prior to Fourier transformation. Chemical shifts were referenced to DSS at 0.00 ppm.
Distance restraints were derived from the 250 ms and 300 ms NOESY spectra recorded at 290 K, 298 K and 300 K. Inter-proton distance restraints were assigned upper-distance bounds of 2.70 Å, 3.50 Å or 5.00 Å corresponding to strong, medium or weak cross-peak volumes, respectively. Pseudoatom corrections were applied where necessary to methylene and methyl protons. Backbone dihedral angle restraints were measured from either 1D NMR spectra or the anti-phase cross-peak splitting in a high digital resolution 2D DQF-COSY spectrum. Stereospecific assignment of methylene protons and χ1 dihedral angle restraints were derived from coupling constants measured from an E-COSY spectrum in combination with HN—Hβ2, HN—Hβ3, Hα—Hα3 and Hα—Hα3 NOE intensities. Slow exchanging amide protons were detected after the sample was lyophilized and reconstituted in 99.99% 2H2O, and were later used to check for consistency of hydrogen bonding interactions in the calculated structures.
Based on the NMR constraint data the three-dimensional structure of Kalata B2 was calculated using a dynamic simulated annealing protocol in the program X-PLOR version 3.1. The procedure was based on that described by Saether et al., 1995 (1). After an initial simulated annealing calculation of a family of 50 structures the ensemble of structures was checked for violations in NOE restraints and ambiguous cross-peaks were resolved on the basis of inter-proton distances. Finally, each member of the ensemble was energy minimized for 1000 cycles using the conjugate gradient Powell algorithm and a refined force field based on the program CHARMm. A schematic representation of the structure of Kalata B2 is shown in FIG. 9 (left panel). The right panel shows an overlay of the structures of Kalata B1 (1) and Kalata B2.
Example 13
Genes Encoding Kalata Like Peptides Belong to a Multigene Family in O. affinis 
Genomic DNA was digested with HindIII, Bam HI, Nde1 and EcoRV and subjected to DNA blot analysis using Oak1 cDNA as probe. About twelve hybridizing bands were obtained in all the digests (FIG. 10) suggesting the cyclotides are derived from a multigene family with up to 12 related genes.
Example 14
Bacterial Expression of the Kalata B1 Precursor Encoded by the Oak1 Clone
The Oak1 cDNA was subcloned into the pQE-30 vector for bacterial expression. A protein of the expected size was induced after addition of IPTG and was purified by immobilized metal affinity chromatography (IMAC) (FIG. 11). This protein has a mass of 12,938±1.2 Da which is consistent with the predicted mass of the protein encoded by the Oak1 clone together with the hexahistidine tag. Furthermore, the six cysteine residues had formed into three disulphide bonds. The metal affinity purified protein produced a single, sharp peak on reversed phase HPLC (FIG. 11B) indicating that the protein had folded to a single conformation.
Example 15
Antibody to the Bacterially Expressed Kalata B1 Precursor Recognizes the Cyclic Peptide
The bacterially expressed Kalata B1 precursor (FIG. 11) which had been purified by metal affinity chromatography and reversed phase HPLC (FIG. 11B) was used to immunize a rabbit to generate polyclonal antibodies. The protein (1 mg/ml) in phosphate buffered saline (11) was emulsified with an equal volume of complete Freund's adjuvant (Gibco/BRL) and 100 μg of protein was injected intramuscularly into a rabbit. The first bleed (15 ml) was taken 14 days after injection and serum was collected after incubation of the blood at 37° C. for 1 hr and at 4° C. overnight. The clotted blood was collected by centrifugation (13000 rpm for 20 min at 4° C.) and serum was collected, divided into 200 μl aliquots and stored at −80° C. A booster injection (prepared as described previously except incomplete Freund's adjuvant was used in place of complete Freund's adjuvant) was administered 4, 8 and 12 weeks after the initial injection. Serum prepared from blood collected two weeks after the third boost was used on the immunoblot shown in FIG. 12 at a 1:1000 dilution.
Purified kalata B1 (1 μg), Kalata B1 precursor (1 μg) and buffer soluble proteins from O. affinis leaves (100 μg) were subjected to SDS-PAGE on a precast 4-12% bis-tris protein gel (Novex, San Diego, Calif., USA) and transferred to a nitrocellulose membrane 0.2 μm pore size) Micron Separations, Inc., Westborough, Mass., USA) in transfer buffer (48 mM Tris-HCl, 192 mM glycine and 20% [w/v] methanol) by using a Novex transfer apparatus at 100V for 1 hr at 4° C. After transfer blots were fixed in isopropanol for 1 min followed by 2% w/v glutaraldehyde for 20 min. The blot was then washed for 5 min in TBS (20 mM Tris-HCl and 150 mM NaCl, pH 7.5) before it was blocked by incubation in 5% (w/v) skim milk powder in TBS for 1 hr at room temperature. The blot was rinsed in TBS for 5 min before incubation for 1 hr at room temperature with the primary antibody diluted (1:1000) in TBST (0.1% [v/v] Tween 20 in TBS) containing 5% w/v skim milk powder. The blot was washed again in TBST before the addition of goat anti-rabbit IgG-HRP conjugate (Amersham, 5×10−3 dilution in TBST) containing 5% w/v skim milk powder. After 1 hr at room temperature the membrane was washed in TBST and bound HRP was detected by the enhanced chemiluminescence (ECL) detection system of Amersham as described by the manufacturer.
The antibody raised to the bacterially expressed precursor protein encoded by Oak1 recognized purified Kalata B1 and the bacterially expressed precursor (FIG. 12). This antibody also bound to a peptide with the same mobility as the Kalata B1 cyclotide in buffer soluble extracts from O. affinis leaves. This antibody has application in location of the Kalata B1 cyclotide in plant cells and as a tool to monitor processing of the precursor and thus to assist in the identification of processing enzymes.
Example 16
Effect of Kalata B1 on the Growth and Development of H. punctigera Larvae
Kalata B1 had a significant effect on the development of larvae. No mortality was observed in the first six days, although 50% failed to survive past day 16 (FIG. 13A). After 16 days none of the 12 survivors had progressed past the first instar stage of development. Most larvae on the control diet, however, had achieved fifth instar (FIGS. 13B, C).
Example 17
Effect of Kalata B1 on the Growth and Development of H. Armigera Larvae
Newly hatched larvae were fed on an artificial diet based on cotton leaves, with and without added Kalata B1. After 12 days on the diet the neonates on the Kalata B1 diet had failed to progress past the first instar stage of development, whereas larvae on the control diet had achieved third to fourth instar. Larvae on the Kalata B1 diet were about 18% of the size of those fed on the control diet.
Example 18
Activity of a Linear Form of Kalata B1 in Insect Bioassays
Kalata peptides isolated from leaves are normally cyclic. The inventors sought to determine whether cyclization was essential for biological activity against insects. Newly hatched H. armigera larvae were added to an artificial diet based on cotton leaves which had been supplemented with cyclic Kalata B1 at either 0.15% w/v of diet or 0.03% w/v of diet. Growth of these larvae was compared to growth of larvae on diet with no added peptide or with added linear Kalata B1 at 0.15% w/v of the diet. Linear Kalata B1 is identical to cyclic Kalata B1 except the backbone chain is open between residues 7 and 8 [see FIG. 1A].
In contrast to cyclic Kalata B1 the linear form of Kalata B1 had no significant effect on growth for the first nine days of the feeding trial. After 12 days larvae fed on the linear peptide were only about 50% the size of control larvae. The cyclic form of Kalata B1 had a more significant effect on growth even when used at one fifth the concentration of the linear form.
Example 19
Cyclotide Genes are Expressed in Roots, Leaves and Stems of O. affinis 
The Oak 1 cDNA was used as a probe on RNA blots containing O. affinis root, shoot and leaf RNA. The Oak 1 clone hybridized to RNA transcripts of about 750 bases which are most abundant in leaves and shoots but are also produced by roots (see FIG. 16).
Example 20
Cyclotide Genes are Conserved Outside the Rubiaceae: Isolation of a Partial cDNA Encoding a Cyclotide from Viola odorata 
Viola odorata produces cyclic peptides that are closely related to Kalata B1 and the other cyclotides produced by O. affinis (12). The inventors sought to determine whether the genes that encode these peptides are conserved in both Viola and Oldenlandia species. Single stranded cDNA was prepared from Viola odorata leaf RNA and cDNA encoding a cyclic peptide was amplified using the procedure and oligonucleotide primers described in Example 3 for O. affinis. A 311 bp fragment was amplified using the Kal2 and oligo-dT-HindIII primers (FIG. 15). This fragment has a 3′ untranslated region of 203 bp and a poly A tail of 18 bp. The predicted peptide encoded by nucleotides 1-78 is identical in sequence to 26 of the 29 amino acid residues of Kalata S, a cyclic peptide that has been extracted from Viola odorata plants (12). The three amino acids not covered by this open reading frame are Gly-Leu-Pro. This indicates that the sequence Gly-Leu-Pro is located at the N-terminus of the mature Kalata sequence in the precursor protein encoded by the full length gene. Thus the potential cleavage site in the predicted V. odorata precursor occurs before the Glycine at the N-terminus and after the asparagine residue at the C-terminus (last residue in the underlined sequence FIG. 15).
The inventors have shown that cyclotides and their encoding genes are conserved in two plant families, Rubiaceae (O. affinis) and Violaceae (V. odorata) that come from two taxonomically distinct subclasses (Asteridae and Rosidae) in the eudicot lineage of flowering plants. This suggests that the cyclotide genes may have arisen early in the evolution of flowering plants and are likely to be conserved in other plant families.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
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## Claims

1. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding a linear precursor form of a cystine knot polypeptide operably linked to a heterologous promoter, wherein said cystine knot polypeptide in its mature form comprises the structure:



wherein C1 to C6 are cysteine residues;
wherein each of C1 and C4, C2 and C5, and C3 and C6 are connected by a disulfide bond to form a cystine knot;
wherein each X represents an amino acid residue in a loop, wherein said amino acid residues are the same or different;
wherein d is about 1-2;
wherein for a, b, c, e, and f, and
i) a is any number from 3-10, and
ii) b, c, e, and f is any number from 1 to 20.

2. The isolated nucleic acid molecule of claim 1, wherein a is from about 3 to 6, b is from about 3 to about 5, c is from about 2 to about 7, e is from about 3 to about 6 and f is from about 4 to about 9.

3. The isolated nucleic acid molecule of claim 1, wherein a is about 3, b is about 4, c is from about 4 to about 7, d is about 1, e is about 4 or 5 and f is from about 4 to about 7.

4. The isolated nucleic acid molecule of claim 1, wherein said linear precursor form of a cystine knot polypeptide encoded by said nucleic acid molecule comprises a plurality of cystine knot polypeptide sequences.

5. The isolated nucleic acid of claim 1, wherein said nucleic acid molecule comprises a nucleotide sequence encoding a linear amino acid sequence of said cystine knot polypeptide flanked by nucleotide sequences encoding amino acid triplets selected from the group consisting of GLP and SLP.

6. The isolated nucleic acid molecule of claim 1, wherein said cystine knot polypeptide is selected from the group consisting of a Kalata B1 polypeptide, a Kalata B2 polypeptide, a Kalata B3 polypeptide, a Kalata B6 polypeptide, and a Kalata B7 polypeptide.

7. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encoding a linear precursor form of a cystine knot polypeptide is from a plant family selected from the group consisting of Rubiaceae and Violaceae.

8. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encoding a linear precursor form of a cystine knot polypeptide is from Oldenlandia affinis DC or Viola odorata.

9. The isolated nucleic acid molecule of claim 1, wherein said sequence of nucleotides encoding a linear precursor form of a cystine knot polypeptide comprises a sequence encoding a signal peptide, wherein said signal peptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 11, 17 and 21.

10. A method for producing a cystine knot polypeptide, comprising: transforming a host cell with a vector comprising the nucleic acid molecule according to claim 1 to express the linear precursor form of a cysteine knot polypeptide, wherein said cystine knot polypeptide is produced.

11. A method for producing a cyclic cystine knot polypeptide, comprising:
a) transforming a host cell with a vector comprising the nucleic acid molecule according to claim 1;
b) expressing said linear precursor form of a cystine knot polypeptide; and
c) processing said linear precursor form of a cystine knot polypeptide to form a cyclic cystine knot polypeptide having the structure:

12. The method of claim 11, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a linear amino acid sequence of said cystine knot polypeptide flanked by nucleotide sequences encoding amino acid triplets that are the same or different and that are selected from the group consisting of GLP and SLP.

13. The method of claim 11 or claim 12, wherein said host cell is a bacterial cell.

14. The method of claim 11 or claim 12, wherein said host cell is a plant cell.

15. The method of claim 11 or claim 12, wherein said plant cell is from a plant family selected from the group consisting of Rubiaceae, Violaceae or Cucurbitaceae.

16. A method for producing a cystine knot polypeptide, comprising:
a) amplifying by polymerase chain reaction a nucleic acid encoding a linear precursor form of a Kalata cyclic polypeptide, said amplifying comprising contacting cDNA encoding a linear precursor form of a Kalata cyclic polypeptide with forward and reverse primers such that said contacting results in amplification of said cDNA to produce an amplified product comprising DNA encoding said linear precursor form of a Kalata cyclic polypeptide, wherein:
i) said forward primer is hybridizable to a sequence complementary to nucleotides 214-231 of SEQ ID NO:4, and
ii) said reverse primer is complementary to nucleotides 272-288 of SEQ ID NO:6,

b) constructing a vector comprising said DNA encoding said linear precursor form of said Kalata cyclic polypeptide, and
c) transforming a host cell with said vector to express the linear Kalata polypeptide, wherein said Kalata cystine knot polypeptide is expressed produced.