Title: Peptides and nucleic
acids derived from Eisenia foetida and the use thereof
United States Patent: 7,060,790
Issued: June 13, 2006
Inventors: de Baetselier;
Patrick (Berchem, BE); Beschin; Alain (Bouffioulx, BE)
Interuniversitair Instituut voor Biotechnologie VZW (Zwijnaarde, BE)
Appl. No.: 596101
Filed: June 16, 2000
Executive MBA in Pharmaceutical Management, U. Colorado
The invention concerns Eisenia foetida
polypeptides and peptides, particularly recombinant polypeptides, which
are useful in tumour therapy, microbial infection, inflammation or
immunology. The invention also relates to a process for preparing the
above-mentioned polypeptides and peptides. Furthermore the invention
concerns nucleic acids coding for said polypeptides and peptides.
OF THE INVENTION
A peptide or polypeptide according to
this invention is characterized by the fact that it contains at least the
13 amino acids as depicted in SEQ ID NO:1 and preferably comprises at
least 9 contiguous amino acids of said SEQ ID NO:1.
To the scope of the invention also relates the polypeptide of SEQ ID NO:3
or functional parts thereof.
According to another embodiment of the invention, the above defined
peptides or polypeptides are exerting a trypanocidal or trypanolytical
activity on T. brucei and/or T. cruzi, alone or preferably in combination
with one of the following characteristics:
exerting cytolytical activity
exhibiting a b-1,3 glucan binding capacity and/or a LPS binding capacity.
exerting opsonizing and/or hemolytic activity
participating in the proPO cascade of E. foetida.
Accordingly another embodiment of the invention is the use of a peptide or
polypeptide comprising at least 9 contiguous amino acids of SEQ ID NO:1,
such as e.g. the polypeptide given by SEQ ID NO:3 for the manufacturing of
a medicament to treat trypanosomal infection, bacterial infection or
cancer. For the treatment of cancer, the peptide or polypeptide can be
linked to a tumour specific antibody that directs the molecule to the
tumor where the (poly)peptide can exert its cytolytical activity.
Another embodiment of the invention is the use of a peptide or polypeptide
comprising at least 9 contiguous amino acids of SEQ ID NO:1 for the
preparation of a medicament to treat a trypanosomal infection, a bacterial
infection or cancer.
The invention thus relates to a DNA sequence encoding an Eisenia foetida
protein or polypeptide or encoding an immunologically active and/or
functional fragment thereof selected from the group consisting of (a) DNA
sequences comprising a nucleotide sequence encoding a protein or peptide
comprising the amino acid sequence as given in SEQ ID NO:1 or 3; (b) DNA
sequences comprising a nucleotide sequence as given in SEQ ID NO:2; (c)
DNA sequences hybridizing with the complementary strand of a DNA sequence
as defined in (a) or (b) and encoding an amino acid sequence which is at
least 80% identical to the amino acid sequence encoded by the DNA sequence
of (a) or (b); (d) DNA sequences the nucleotide sequence of which is
degenerated as a result of the genetic code to a nucleotide sequence of a
DNA sequence as defined in any one of (a) to (c); and (e) DNA sequences
encoding a fragment of a protein encoded by a DNA sequence of any one of
(a) to (d).
The present invention also relates to vectors, particularly plasmids,
cosmids, viruses, bacteriophages and other vectors used conventionally in
genetic engineering that contain a nucleic acid molecule alternatively
called nucleic acid sequence or DNA sequence according to the invention.
Methods which are well known to those skilled in the art can be used to
construct various plasmids and vectors; see, for example, the techniques
described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring
Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular
Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989).
Alternatively, the nucleic acid molecules and vectors of the invention can
be reconstituted into liposomes for delivery to target cells.
In a preferred embodiment the nucleic acid molecule present in the vector
is linked to (a) control sequence(s) which allow the expression of the
nucleic acid molecule in prokaryotic and/or eukaryotic cells.
The term "control sequence" refers to regulatory DNA sequences which are
necessary to effect the expression of coding sequences to which they are
ligated. The nature of such control sequences differs depending upon the
host organism. In prokaryotes, control sequences generally include
promoter, ribosomal binding site, and terminators. In eukaryotes generally
control sequences include promoters, terminators and, in some instances,
enhancers, transactivators or transcription factors. The term "control
sequence" is intended to include, at a minimum, all components the
presence of which are necessary for expression, and may also include
additional advantageous components.
The term "operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to function
in their intended manner. A control sequence "operably linked" to a coding
sequence is ligated in such a way that expression of the coding sequence
is achieved under conditions compatible with the control sequences. In
case the control sequence is a promoter, it is obvious for a skilled
person that double-stranded nucleic acid is used.
Thus, the vector of the invention is preferably an expression vector. An
"expression vector" is a construct that can be used to transform a
selected host cell and provides for expression of a coding sequence in the
selected host. Expression vectors can for instance be cloning vectors,
binary vectors or integrating vectors. Expression comprises transcription
of the nucleic acid molecule preferably into a translatable mRNA.
Regulatory elements ensuring expression in prokaryotic and/or eukaryotic
cells are well known to those skilled in the art. In the case of
eukaryotic cells they comprise normally promoters ensuring initiation of
transcription and optionally poly-A signals ensuring termination of
transcription and stabilization of the transcript. Additional regulatory
elements may include transcriptional as well as translational enhancers.
Possible regulatory elements permitting expression in prokaryotic host
cells comprise, e.g., the P.sub.L, lac, trp or tac promoter in E. coli,
and examples of regulatory elements permitting expression in eukaryotic
host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-,
RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin
intron in mammalian and other animal cells. In this context, suitable
expression vectors are known in the art such as Okayama-Berg cDNA
expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene),
pSPORT1 (GIBCO BRL). Advantageously, the above-described vectors of the
invention comprises a selectable and/or scorable marker. Selectable marker
genes are well known to those skilled in the art and comprise, for
example, npt which confers resistance to the aminoglycosides neomycin,
kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987 995) and
hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481
Useful scorable marker are also known to those skilled in the art and are
commercially available. Advantageously, said marker is a gene encoding
luciferase (Giacomin, Pl. Sci. 116 (1996), 59 72; Scikantha, J. Bact. 178
(1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44
47) or .beta.-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901 3907). This
embodiment is particularly useful for simple and rapid screening of cells,
tissues and organisms containing a vector of the invention.
The present invention furthermore relates to host cells comprising a
vector as described above or a nucleic acid molecule according to the
invention wherein the nucleic acid molecule is foreign to the host cell.
By "foreign" it is meant that the nucleic acid molecule is either
heterologous with respect to the host cell, this means derived from a cell
or organism with a different genomic background, or is homologous with
respect to the host cell but located in a different genomic environment
than the naturally occurring counterpart of said nucleic acid molecule.
This means that, if the nucleic acid molecule is homologous with respect
to the host cell, it is not located in its natural location in the genome
of said host cell, in particular it is surrounded by different genes. In
this case the nucleic acid molecule may be either under the control of its
own promoter or under the control of a heterologous promoter. The vector
or nucleic acid molecule according to the invention which is present in
the host cell may either be integrated into the genome of the host cell or
it may be maintained in some form extrachromosomally.
The host cell can be any prokaryotic or eukaryotic cell, such as
bacterial, insect, fungal, plant or animal cells. Preferred fungal cells
are, for example, those of the genus Saccharomyces, in particular those of
the species S. cerevisiae.
Another subject of the invention is a method for the preparation of an
Eisenia foetida protein which comprises the cultivation of host cells
according to the invention which, due to the presence of a vector or a
nucleic acid molecule according to the invention, are able to express such
a protein, under conditions which allow expression of the protein and
recovering of the so-produced protein from the culture.
The term "expression" means the production of a protein or nucleotide
sequence in the cell. However, said term also includes expression of the
protein in a cell-free system. It includes transcription into an RNA
product, post-transcriptional modification and/or translation to a protein
product or polypeptide from a DNA encoding that product, as well as
possible post-translational modifications. Depending on the specific
constructs and conditions used, the protein may be recovered from the
cells, from the culture medium or from both. For the person skilled in the
art it is well known that it is not only possible to express a native
protein but also to express the protein as fusion polypeptides or to add
signal sequences directing the protein to specific compartments of the
host cell, e.g., ensuring secretion of the peptide into the culture
medium, etc. Furthermore, such a protein and fragments thereof can be
chemically synthesized and/or modified according to standard methods.
The terms "protein" and "(poly)peptide" used in this application are
interchangeable. "(Poly)peptide" refers to a polymer of amino acids (amino
acid sequence) and does not refer to a specific length of the molecule.
Thus peptides and oligopeptides are included within the definition of
polypeptide. This term does also refer to or include post-translational
modifications of the polypeptide, for example, glycosylations,
acetylations, phosphorylations and the like. Included within the
definition are, for example, polypeptides containing one or more analogs
of an amino acid (including, for example, unnatural amino acids, etc.),
polypeptides with substituted linkages, as well as other modifications
known in the art, both naturally occurring and non-naturally occurring.
The present invention furthermore relates to proteins encoded by the
nucleic acid molecules according to the invention or produced or obtained
by the above-described methods, and to functional and/or immunologically
active fragments of such proteins. The proteins and polypeptides of the
present invention are not necessarily translated from a designated nucleic
acid sequence; the polypeptides may be generated in any manner, including
for example, chemical synthesis, or expression of a recombinant expression
system, or isolation from a suitable viral system. The polypeptides may
include one or more analogs of amino acids, phosphorylated amino acids or
unnatural amino acids. Methods of inserting analogs of amino acids into a
sequence are known in the art. The polypeptides may also include one or
more labels, which are known to those skilled in the art. In this context,
it is also understood that the proteins according to the invention may be
further modified by conventional methods known in the art. By providing
the proteins according to the present invention it is also possible to
determine fragments which retain biological activity, namely the mature,
processed form. This allows the construction of chimeric proteins and
peptides comprising an amino sequence derived from the protein of the
invention, which is crucial for its binding activity and other functional
amino acid sequences. The other functional amino acid sequences may be
either physically linked by, e.g., chemical means to the proteins of the
invention or may be fused by recombinant DNA techniques well known in the
The term "fragment of a sequence" or "part of a sequence" means a
truncated sequence of the original sequence referred to. The truncated
sequence (nucleic acid or protein sequence) can vary widely in length; the
minimum size being a sequence of sufficient size to provide a sequence
with at least a comparable function and/or activity of the original
sequence referred to, while the maximum size is not critical. In some
applications, the maximum size usually is not substantially greater than
that required to provide the desired activity and/or function(s) of the
original sequence. Typically, the truncated amino acid sequence will range
from about 5 to about 60 amino acids in length. More typically, however,
the sequence will be a maximum of about 50 amino acids in length,
preferably a maximum of about 30 amino acids. It is usually desirable to
select sequences of at least about 10, 12 or 15 amino acids, up to a
maximum of about 20 or 25 amino acids. Specifically in this context is
referred to SEQ ID NO:1 comprising 13 amino acids showing the essential
cytolytic, trypanolytic and glucan-binding characteristics comparable to
the whole protein.
Furthermore, folding simulations and computer redesign of structural
motifs of the protein of the invention can be performed using appropriate
computer programs (Olszewski, Proteins 25 (1996), 286 299; Hoffman, Comput.
Appl. Biosci. 11 (1995), 675 679). Computer modeling of protein folding
can be used for the conformational and energetic analysis of detailed
peptide and protein models (Monge, J. Mol. Biol. 247 (1995), 995 1012;
Renouf, Adv. Exp. Med. Biol. 376 (1995), 37 45). In particular, the
appropriate programs can be used for the identification of interactive
sites of the protein according to the invention, its receptor, its ligand
or other interacting proteins by computer assistant searches for
complementary peptide sequences (Fassina, Immunomethods 5 (1994), 114
120). Further appropriate computer systems for the design of protein and
peptides are described in the prior art, for example in Berry, Biochem.
Soc. Trans. 22 (1994), 1033 1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987),
1 13; Pabo, Biochemistry 25 (1986), 5987 5991. The results obtained from
the above-described computer analysis can be used for, e.g., the
preparation of peptidomimetics of the protein of the invention or
fragments thereof. Such pseudopeptide analogues of the natural amino acid
sequence of the protein may very efficiently mimic the parent protein (Benkirane,
J. Biol. Chem. 271 (1996), 33218 33224). For example, incorporation of
easily available achiral-amino acid residues into a protein of the
invention or a fragment thereof results in the substitution of amide bonds
by polymethylene units of an aliphatic chain, thereby providing a
convenient strategy for constructing a peptidomimetic (Banerjee,
Biopolymers 39 (1996), 769 777). Superactive peptidomimetic analogues of
small peptide hormones in other systems are described in the prior art
(Zhang, Biochem. Biophys. Res. Commun. 224 (1996), 327 331). Appropriate
peptidomimetics of the protein of the present invention can also be
identified by the synthesis of peptidomimetic combinatorial libraries
through successive amide alkylation and testing the resulting compounds,
e.g., for their binding and immunological properties. Methods for the
generation and use of peptidomimetic combinatorial libraries are described
in the prior art, for example in Ostresh, Methods in Enzymology 267
(1996),220 234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709 715.
Furthermore, a three-dimensional and/or crystallographic structure of the
protein of the invention can be used for the design of peptidomimetic
inhibitors of the biological activity of the protein of the invention
(Rose, Biochemistry 35 (1996), 12933 12944; Rutenber, Bioorg. Med. Chem. 4
(1996), 1545 1558).
Furthermore, the present invention relates to antibodies specifically
recognizing an Eisenia foetida protein according to the invention or
parts, i.e. specific fragments or epitopes, of such a protein. The
antibodies of the invention can be used to identify and isolate other
Eisenia foetida proteins and genes in any organism. These antibodies can
be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as
well as fragments of antibodies, such as Fab, Fv or scFv fragments etc.
Monoclonal antibodies can be prepared, for example, by the techniques as
originally described in Kohler and Milstein, Nature 256 (1975), 495, and
Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse
myeloma cells to spleen cells derived from immunized mammals. Furthermore,
antibodies or fragments thereof to the aforementioned peptides can be
obtained by using methods which are described, e.g., in Harlow and Lane
"Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
These antibodies can be used, for example, for the immunoprecipitation and
immunolocalization of proteins according to the invention as well as for
the monitoring of the synthesis of such proteins, for example, in
recombinant organisms, and for the identification of compounds interacting
with the protein according to the invention. For example, surface plasmon
resonance as employed in the BIAcore system can be used to increase the
efficiency of phage antibodies selections, yielding a high increment of
affinity from a single library of phage antibodies which bind to an
epitope of the protein of the invention (Schier, Human Antibodies
Hybridomas 7 (1996), 97 105; Malmborg, J. Immunol. Methods 183 (1995),7
13). In many cases, the binding phenomena of antibodies to antigens is
equivalent to other ligand/anti-ligand binding.
Claim 1 of 2 Claims
1. An isolated peptide
consisting of SEQ ID NO: 1.
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