Note: Descriptions are shown in the official language in which they were submitted.
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Nucleic acid molecules encoding an amylosucrase
The present invention relates to nucleic acid molecules encoding a protein
having
amylosucrase activity and to vectors containing such molecules. Furthermore,
the
invention relates to the production of a-1,4 glucans and fructose using the
described
nucleic acid molecules or the encoded proteins.
Linear a-1,4 glucans are polysaccharides consisting of glucose monomers, the
latter
being exclusively linked to each other by a-1,4 glycosidic bonds. The most
frequently
occurring natural a-1,4 glucan is amylose, a component of plant siarch.
Recently,
more and more importance has been attached to the commercial use of linear a-
1,4
glucans. Due to its physico-chemical properties amylose can be used to produce
films that are colorless, odorless and flavorless, non-toxic and biologically
degradable. Already today, there are various possibilities of application,
e.g., in the
food industry, the textile industry, the glass fiber industry and in the
production of
paper.
One has also succeeded in producing fibers from amylose whose properties are
similar to those of natural cellulose fibers and which allow to partially or
even
completely replace them in the production of paper. Being the most important
representative of the linear cx-1,4 glucans, amylose is particularly used as
binder for
the production of tablets, as thickener of puddings and creams, as gelatin
substitute,
as binder in the production of sound-insulating wall panels and to improve the
flow
properties of waxy oils. Another property of the a-1,4 glucans, wi~ich
recently has
gained increasing attention, is the capability of these molecules to form
inclusion
compounds with organic complexers due to their helical structure. This
property
allows to use the a-1,4 glucans for a wide variety of applications. Present
considerations relate to their use for the molecular encapsulation of
vitamins,
pharmaceutical compounds and aromatic substances, as well as their use for the
chromatographic separation of mixtures of substances over immobilized linear a-
1,4
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glucans. Amylose also serves as starting material for the production of so-
called
cyclodextrins (also referred to as cycloamyloses, cyclomaltoses) which in turn
are
widely used in the pharmaceutical industry, food processing technology,
cosmetic
industry and analytic separation technology. These cyclodextrins are cyclic
maltooligosaccharides from 6-8 monosaccharide units, which are freely soluble
in
water but have a hydrophobic cavity which can be utilized to form inclusion
compounds.
Today, a-1,4 glucans, in particular linear a-1,4 glucans, are obtained in the
form of
amylose from starch. Starch itself consists of two components. One component
forms
the amylose as an unbranched chain of a-1,4 linked glucose units. The other
component for ms the amylopectin, a highly branched polymer from glucose units
in
which in addition to the a-1,~4 links the glucose chains can also be branched
via a-1,6
links. Due to their different structure and the resulting physico-chemical
properties,
the two components are also used for different fields of application. In order
to be
able to directly utilize the properties of the individual components, it is
necessary to
obtain them in pure form. Both components can be obtained from starch, the
process, however, requiring several purification steps and involving
considerable time
and money. Therefore, there is a need to find possibilities of obtaining both
components of the starch in a uniform manner. It is known that certain
bacteria, in
particular those of the genus Neisseria produce enzymes capab~~ of
synthesizing
linear a-1,4 glucans from sucrose. In order to be able to use such enzymes for
the
efficient production of a-1,4 glucans, it is necessary to isolate and
characterize the
corresponding DNA sequences.
The technical problem underlying the present invention is therefore to provide
nucleic
acid molecules and processes that allow the production of a-1,4 glucans.
The solution of this technical problem is achieved by the present invention by
providing the embodiments characterized in the claims.
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The invention therefore relates to nucleic acid molecules encoding a protein
having
the enzymatic activity of an amylosucrase selected from the group consisting
of
(a) nucleic acid molecules encoding a protein comprising the amino acid
sequence as depicted in SEQ ID NO: 2;
(b) nucleic acid molecules comprising the nucleotide sequence of the coding
region as indicated in SEQ ID NO: 1;
(c) nucleic acid molecules encoding an analogue of the polypeptide having the
amino acid sequence as depicted under SEQ lD NO: 2; and
(d) nucleic acid molecules, the sequence of which differs from the sequence of
a
nucleic acid molecule as defined in (c) due to the degeneracy of the genetic
code.
The nucleic acid sequence of the coding region depicted in SEQ ID NO: 1
encodes a
protein of Neisseria polysaccharea having the enzymatic activity of an
amylosucrase.
With the help of the nucleic acid molecules of the present invention it is
possible to
produce microorganisms and fungi, particularly yeasts, that are capable of
producing
an enzyme catalyzing the synthesis of a-1,4 glucans from sucrose.
It is furthermore possible to produce at low production costs a-1,4 glucans,
in
particular linear a-1,4 glucans, as well as pure fructose syrup with the help
of the
DNA sequences of the invention or of the proteins encoded by them.
Nucleotide sequences which encodes an analogue of the polypeptide as depicted
in
SEQ ID NO: 2 are understood in the scope of the present invention as
nucleotide
sequence which encode a palypeptide having the following characteristics:
(a) it has amylosucrase activity; and preferably,
(b) it furthermore shows an identity on the amine acid sequence level of at
least
80%, more preferably of at least 85%, even more preferably of at least 90%
and particularly preferred of at least 95%, to the amino acid sequence as
depicted in SEQ ID NO: 2 over its complete length.
Thus, the present invention also relates to nucleic acid molecules encoding a
polypeptide the sequence of which differs at one or more positions from the
amino
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acid sequence as depicted in SEQ lD NO: 2 and which still has amylosucrase
activity. The differences in the amino acid sequence may be due to
replacements of
amino acid residues by other amino acid residues, to the addition of amino
acid
residues, preferably at the N- or C-terminus of the polypeptide, or to
deletions of one
or more amino acid residues, preferably at the N- or C-terminus of the
protein. The
generation of nucleic acid molecules encoding such analogues of the described
protein is well within the common general knowledge of the person skilled in
the art.
The present invention also relates to nucleic acid molecules the complementary
strand of which hybridizes under stringent conditions to a nucleic acid
molecule as
defined above and which encode a polypeptide having the enzymatic activity of
an
amylosucrase.
In this invention the term "hybridization" means a hybridization under
stringent
conditions as described for example in Sambrook et al., Molecular Cloning, A
Laboratory Manual, 2"d Edition (1989) Cold Spring Harbor Laboratory Press,
Cold
Spring Harbor, NY). "Stringent conditions" mean that there is a sequence
identity of
at least 80% of the completes coding sequence, preferably an identity of at
least 90%,
more preferably of at least 9~% and particularly preferred of at least 99%.
Nucleic acid molecules hybridizing to the molecules according to the invention
may
be isolated e.g. from genomic or from cDNA libraries produced from organism
expressing an amylosucrase, for example, from microorganisms, in particular
from
bacteria of the genus Neisseria. The identification and isolation of such
nucleic acid
molecules may take place by using the molecules according to the invention or
parts
of these molecules or, as the case may be, the reverse complement strands of
these
molecules, e.g. by hybridization according to standard methods (see e.g.
Sambrook
et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY).
As a probe for hybridization e.g. nucleic acid molecules may be used which
exactly or
basically contain the nucleotide sequence of the coding region indicated under
SEQ
ID NO. 1 or parts thereof. The fragments used as hybridization probe may also
be
synthetic fragments which were produced by means of the conventional
synthesizing
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methods and the sequence of which is basically identical with that of a
nucleic acid
molecule according to the invention. After identifying and isolating the genes
hybridizing to the nucleic acid sequences according to the invention, the
sequence
has to be determined and the properties of the proteins encoded by this
sequence
have to be analyzed.
The molecules hybridizing to the nucleic acid molecules of the invention also
comprise fragments, derivatives and allelic variants of the above-described
nucleic
acid molecules which encode a protein having the enzymatic activity of an
amylosucrase. Thereby, fragments are defined as parts of the nucleic acid
molecules, which are long enough in order to encode a protein still having the
enzymatic activity. This includes also parts of nucleic acid molecules
according to the
invention which lack the nucleotide sequence encoding the signal peptide
responsible for the secretion of the protein. The term derivatives means that
the
sequences of these molecules differ from the sequences of the above-mentioned
nucleic acid molecules at onE: or mare positions and that they exhibit a high
degree of
homology to these sequences. Hereby, homology means a sequence identity of at
least 80%, in particular an identity of at least 90%, preferably of more than
95% and
still more preferably a sequence identity of more than 98%. The deviations
occurring
when comparing with the above-described nucleic acid molecules might have been
caused by deletion, substitution, insertion or recombination.
Moreover, homology means that functional and/or structural equivalence exists
between the respective nucleic acid molecules or the proteins they encode. The
nucleic acid molecules, which are homologous to the above-described molecules
and
represent derivatives of these molecules, are generally variations of these
molecules,
that constitute modifications which exert the same biological function. These
variations may be naturally occurring variations, for example sequences
derived from
other organisms, or mutations, whereby these mutations may have occurred
naturally
or they may have been introduced by means of a specific mutagenesis. Moreover
the
variations may be synthetically produced sequences. The allelic variants may
be
naturally occurring as well as synthetically produced variants or variants
produced by
recombinant DNA techniques.
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The proteins encoded by the various variants of the nucleic acid molecules
according
to the invention exhibit certain common characteristics. Enzyme activity,
molecular
weight, immunologic reactivity, conformation etc. may belong to these
characteristics
as well as physical properties such as the mobility in gel electrophoresis,
chromatographic characteristics, sedimentation coefficients, solubility,
spectroscopic
properties, stability, pH-optimum, temperature-optimum etc.
An amylosucrase (also referred to as sucrose:l,4-a glucan 4-a-
glucosyltransferase,
E.C. 2.4.1.4.) is an enzyme for which the following reaction scheme is
suggested:
sucrose + (a-1,4-D-glucosyl)c ~ D-fructose + (a-1,4-D-glucosyl)n+1
This reaction is a transglucosylation. The transglucosylation can take place
in the
presence or absence of acceptor molecules. Such acceptor molecules can be
polysaccharides, such as maltooligosaccharides, dextrin, glycogen etc. When
such
an acceptor molecule is a linear, aligomeric a-1,4-glucan, the resulting
product is a
polymeric linear a-1,4-gluran. When the transglucosylation catalyzed by the
amylosucrase is carried out in the absence of such acceptor molPr_ule, a
glucan is
obtained which comprises a terminal fructose molecule. All the products
obtainable
by transglycosylation with the help of an amylosucrase in the absence or
presence of
an acceptor molecule are referred to in the scope of the present i7vention as
a-1,4
glucans.
The reaction mechanism far a transglucosylation by an amylosucrase in the
absence
of an acceptor molecule can be described as follows:
G-F+n(G-F) --~ G~-G-F+nF,
wherein G-F is sucrose. G is glucose, F is fructose and G"-G-F is an a-1.,4
glucan.
The reaction mechanism in the presence of an acceptor molecule can be
described
as follows:
mG-F+G~ -~ G~+m-~mF.
wherein G~ is a polysaccharide acceptor molecule, G~+", is the polysaccharide
plus a-
1,4 glucan chains added thereto by amylosucrase, G-F is sucrose, F is fructose
and
G is glucose. The products of the reaction catalyzed by an amylosucrase are
the
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above described a-1,4 glucans and fructose. Cofactors are not required.
Amylosucrase activity so far has been found only in few bacteria species,
among
them particularly the species Neisseria (MacKenzie et al., Can. J. Microbiol.
24
(1978), 357-362) and the enzyme has been examined only for its enzymatic
activity.
According to Okada et al., the partially purified enzyme from Neisseria
perflava upon
addition of sucrose results in the synthesis of glycogen-like polysaccharides
which
are branched to a small extent (Okada et al., J. Biol. Chem. 249 (1974), 126-
135).
Likewise, the intra- or extracellularly synthesized glucans of Neisseria
perflava and
Neisseria polysaccharea exhibit a certain degree of branching (Riou et al.,
Can. J.
Microbiol. 32 (1986), 909-911). Whether these branches are introduced by the
amylosucrase or via another enzyme that is present in the purified
amylosucrase
preparations as contamination, has so far not been elucidated. Since an enzyme
introducing branching has so far not been found, it is assumed that both the
polymerization and the branching reactions are catalyzed by amylosucrase
(Okada et
ai., ioc. cit.).
The enzyme that is expressed in a constitutive manner in Neisseria is
extremely
stable, binds very strongly to the polymerization products and is
competitively
inhibited by the product fructose (MacKenzie et al., Can. J. Microbiol. 23
(1977),
1303-1307). The Neisseria species Neisseria polysaccharea secretes the
amylosucrase (Riou et al., loc. cit.) while in the other Neisseria species it
remains in
the cell. Enzymes having amylosucrase activity could only be detected in
microorganisms. Plants are not known to have amylosucrases.
The detection of the enzymatic activity of the amylosucrase can he achieved by
detecting the synthesized glucans, as is described in Example 3, below.
Detection is
usually carried out by using a iodine stain. It is possible to identify
bacterial colonies
expressing amylosucrase by, e.g,, treatment with iodine vapor. Colonies
synthesizing
the a-1,4 glucans are stained blue.
The enzyme activity of the purified enzyme can be detected on, e.g., sucrose-
containing agarose plates. If the protein is applied to such a plate and
incubated for
about 1 h or more at 37°C, it diffuses into the agarose and catalyzes
the synthesis of
glucans. The latter can be detected by treatment with iodine vapor.
Furthermore, the
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protein can be detected in native polyacrylamide gels. After a native
palyacrylamide
gel electrophoresis, the gel is equilibrated in sodium citrate buffer (50 mM,
pH 6.5)
and incubated over night in a sucrose solution (5% in sodium citrate Duffer).
If the gel
is subsequently stained with Lugol's solution, areas in which proteins having
amylosucrase activity are iacalized are stained blue due to the synthesis of a-
1,4
glucans.
The protein encoded by a nucleic acid molecule according to the invention
preferably
has a molecular weight of 63t 20kDa, more preferably of 63t 15kDa and even
more
preferably of 63t 10kDa when determined in an SDS-PAGE.
In a preferred embodiment, the invention relates to nucleic acid molecules
encoding
an amylosucrase from a microorganism, particularly a gram negative
microorganism,
preferably from a bacterium of the species Neisseria and particularly
preferred from
Neisseria polysaccharea.
The nucleic acid molecules according to the invention can be any kind if
nucleic acid
molecule, for example, RNA. or DNA, in particular cDNA or genomic DNA. They
can
be synthetic, partly synthetic or isolated from natural sources.
Furthermore, the present invention relates to vectors, for example, plasmids,
phages,
cosmids, phagemids or artificial chromosomes, containing a nuch:ic acid
molecule
according to the invention. The invention particularly relates to vectors in
which the
nucleic acid molecule of the invention is linked to sequences ensuring
expression of
the nucleic acid molecule in prokaryotic or eukaryotic host cells. Expression
in this
regard means transcription, preferably transcription and translation.
Expression
vectors have been extensively described in the art. fn addition to a selection
marker
gene and a replication origin allowing replication in the selected host they
normally
contain a promoter active in the host cell and a transcription termination
signal.
Between promoter and termination signal there is normally at least one
restriction site
or one polylinker which allaws insertion of a coding DNA sequence. As promoter
sequence the DNA sequence which normally controls trans~.ription of the
corresponding gene can be used as long as it is active in the selected
organism. This
sequence can be replaced by other promoter sequences. Promoters can be used
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that effect constitutive expression of the gene or inducible promoters that
allow a
selective regulation of the expression of the gene downstream thereof.
Bacterial and
viral promoter sequences for the expression in prokaryotic host cells have
been
extensively described in the art. Promoters allowing a particularly strong
expression
of the gene downstream thereof, are, e.g., the T7 promoter (Studier et al., in
Methods
in Enzymology 185 (1990), 60-89), lacuv5, trp, trp-IacUVS (DeBoer et al., in
Rodriguez, R.L. and Chamberlin, M.J., (Eds.), Promoters, Structure and
Function;
Praeger, New York, 1982, pp. 462-481; DeBoer et al., Proc. Natl. Acad. Sci.
USA 80
(1983), 21-25), Ipl, rac (Boros et al., Gene 42 (1986), 97-100) or the ompF
promoter.
Vectors for the expression of heterologous genes in yeasts have also been
described
(e.g., Bitter et al., Methods in Enzymology 153 (1987), 516-544). These
vectors, in
addition to a selection marker gene and a replication origin for the
propagation in
bacteria, contain at least one further selection marker gene that allows
identification
of transformed yeast cells, a DNA sequence allowing replication in yeasts and
a
polyiinker for the insertion of the desired expression cassette. The
expression
cassette is constructed from promoter, DNA sequence to be expressed and a DNA
sequence allowing transcriptional termination and polyadenylation of the
transcript.
Promoters and transcriptional termination signals from Saccharomyces have also
been described and are available. An expression vector can be introduced into
yeast
cells by transformation according to standard techniques (Methods in Yeast
Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press,
1990).
Cells containing the vector are selected and propagated on apprc:priate
selection
media. Yeasts furthermore allow to integrate the exFression cassette via
homologous
recombination into the genome of a cell using an appropriate vector, leading
to a
stable inheritance of the feature.
Furthermore, the present invention relates to host cells transformed with a
nucleic
acid molecule or with a vector according to the invention. Suitable host cells
are
prokaryotic cells, such as microorganisms, e.g. bacteria, such as E. coli,
Bacillus,
Streptococcus etc., or eukaryotic cells, e.g. fungal cells, such as
Saccharornyces
cerevisiae; plant cells or animal cells, e.g. insect cells, CHO cells etc.
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Moreover, the present invention relates to a process for producinn ;a protein
having
amyiosucrase activity comprising culturing a host cell according to the
invention
under conditions allowing expression of the protein and recovering the protein
from
the cells and/or the culture medium.
The present invention also relates to a protein having the enzymatic activity
of an
amylosucrase which is encoded by a nucleic acid molecule according to the
invention, or which is obtainable by the process according to the invention.
In another aspect the present invention relates to a process for producing a-
1,4
glucans and/or fructose comprising
(a) culturing a host cell according to the invention which secrets the
amylosucrase
into the culture medium in a medium which contains sucrose and under
conditions allowing expression and secretion of the amylosucr=_~se; and
(b) recovering the produced a-1,4 glucans andior the fructose from the culture
medium.
The above described process now allows to produce pure a-1,4 glucans in vitro.
The
amylosucrase expressed by Neisseria polysaccf~area is an extracelluiar enzyme
which synthesizes linear a-'k ,4 glucans outside of the cells on the basis of
sucrose.
Unlike in the most pathways of synthesis for polysaccharides that proceed
within the
cell, neither activated glucose derivatives nor cofactors are required. The
energy that
is required for the formation of the a-1,4 glucosidic link between the
condensed
glucose residues is directly obtained from the hydrolysis of the link between
the
glucose and the fructose unit in the sucrose molecule.
It is therefore possible to cultivate amylosucrase-secreting host cells in a
sucrose-
containing medium, with the secreted amylosucrase leading to a synthesis of a-
1,4
glucans from sucrose in the medium. These glucans can be isolate) from the
culture
medium.
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Furthermore, the process according to the invention allows to produce in an
inexpensive manner pure fructose syrup. Conventional methods for the
production of
fructose either contemplate the enzymatic hydrolysis of sucrose using an
invertase or
the degradation of starch into glucose units, often by acidolysis, and
subsequent
enzymatic conversion of the glucose into fructose by glucose isomerase. Both
methods result in mixtures of glucose and fructose. The two components have to
be
separated from each other by chromatographic processes which are time
consuming
and expensive.
In the process according of the invention, the separation of the substrate,
sucrose,
from the two reaction products, fructose and a-1,4 glucans, or separation of
the two
reaction products can be achieved by, e.g., using membranes allowing the
permeation of fructose but not of sucrose or glucans. If the fructose is
continuously
removed via such a membrane, the sucrose is converted more or less completely
into fructose and linear glucans.
Also the amylosucrase producing cells can preferably be immobilized on a
carrier
material located between two membranes, one of which allows the permeation of
fructose but not of sucrose or glucans and the other allows the permeation of
sucrose
but not of glucans. The substrate is supplied through the membrane which
allows the
permeation of sucrose. The synthesized glucans remain in the space between the
two membranes and the released fructose can continuously be removed from the
reaction equilibrium through the membrane which allows only the: permeation of
fructose. Such a set-up allows an efficient separation of the reaction
products and
thus inter olio the production of pure fructose.
The use of amylosucrases for the production of pure fructose offers the
advantage
that the comparably inexpensive substrate sucrose can be used as starting
material
and furthermore that the fructose can be isolated from the reaction mixture in
a
simple manner without chromatographic processes.
In a preferred embodiment the host cells used in the process is a
microorganism,
such as Saccharomyces cerevisiae or E coli, and even more preferably the host
cell
is immobilized. Immobilization generally is achieved by inclusion of the cells
in an
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appropriate material such as, e.g., alginate, polyacrylamide, ge'a;in,
cellulose or
chitosan. It is, however, also possible to adsorb or covalently bind the cells
to a
carrier material (Brodelius and Mosbach, in Methods in Enzymology, Vol.
135:173-
175). An advantage of the immobilization of cells is that considerably higher
cell
densities can be achieved than by cultivation in a liquid culture, resulting
in a higher
productivity. Also the costs for agitation and ventilation of the culture as
welt as for
the measures for maintaining sterility are reduced. An important aspect is
that
immobilization allows a continuous production so that long unproductive phases
which usually occur in fermentation processes can be avoided or can at least
be
considerably reduced. As mentioned above, yeast cells expressing an
amylosucrase
can be used as a microorganism in the process. Cultivation methods for yeasts
have
been sufficiently described (Methods in Yeast Genetics, A Laboratory Course
Manual, Cold Spring Harbor Laboratory Press, 1990). Immobilization of the
yeasts is
also possible and is already used in the commercial prod~~;:;~on of ethanol
(Nagashima et al., in Methods in Enzymology 136, 394-405; Nojima and Yamada,
in
Methods in Enzymology 136, 380-394).
However, the use of yeasts secreting amylosucrase for the synthesis of a-1,4
glucans in sucrose-containing media is not readily possible as yeasts secrete
an
invertase that hydrolyzes extracellular sucrose. The yeasts import the
resulting
hexoses via a hexose transporter. Gozalbo and Hohmann (Current Genetics 17
(1990), 77-79), however, describe a yeast strain that carries a defective suc2
gene
and that therefore cannot secrete invertase. Also, these yeast cells do not
contain a
transport system for importing sucrose into the cells. If such a strain is
modified with
the nucleic acid molecule of the invention such that it secretes an
amylosucrase into
the culture medium, a-1,4 glucans are synthesized by the amylosucrase if the
culture
medium contains sucrose. The fructose being formed as reaction product may
subsequently be imported by the yeasts.
Furthermore, the present invention relates to a process for the production of
a-1,4
glucans and/or fructose in vitro comprising the step of bringing a protein
according to
the invention into contact with a sucrose-containing solution under conditions
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allowing the conversion of sucrose to a-1,4 glucans and fructose and
recovering the
produced a-1,4 glucans and/or fructose from the solution.
In particular, it is possible to synthesize a-1,4 glucans in vitro with the
help of a cell-
free enzyme preparation. This may be obtained, for example, by cultivating
amylosucrase-secreting host cells in a sucrose-free medium allowing expression
of
the amylosucrase until the tationary growth phase is reached. After removal of
the
cells from the growth medium by centrifugation the secreted enzyme: can be
obtained
from the supernatant. The enzyme can then be added to sucrose-containing
solutions to synthesize a-1,4 glucans and fructose. As compared to the
synthesis of
a-1,4 glucans directly in a sucrose-containing growth medium this method is
advantageous in that the reaction conditions can be better controlled and that
the
reaction products are substantially purer and can more easily be further
purified.
The enzyme can be purified from the culture medium by conventional
purification
techniques such as precipitation, ion exchange chromatography, affinity
chromatography, gel filtration, HP~C reverse phase chromatography, etc.
It is furthermore possible to express a polypeptide by modification of the DNA
sequence inserted into the expression vector leading to a polypeptide which
can be
isolated more easily from the culture medium due to certain properties. It is
possible
to express the enzyme as a fusion protein along with another polypeptide
sequence
whose specific binding properties allow isolation of the fusion protein via
affinity
chromatography.
Known techniques are, e.g., expression as fusion protein along with glutathion
S
transferase and subsequent purification via affinity chromatography on a
glutathion
column, making use of the affinity of the glutathion S transferase to
glutathion (Smith
and Johnson, Gene 67 (1988), 31-40). Another known technique is the expression
as
fusion protein along with the maltose binding protein (MBP) and subsequent
purification on an amylose column (Guan et al., Gene 67 (1988), 21-30; Mains
et al.,
Gene 74 (1988), 365-373)
In a preferred embodiment, the amylosucrase in such a process is immobilized.
In addition to the possibilit~,r of directly adding the purified enzyme to a
sucrose-
containing solution to synthesize a-1,4 glucans, there is the alternative of
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immobilizing the enzyme on a carrier material. Such immobilization offers the
advantage that the enzyme as synthesis catalyst can easily be retrieved and
can be
used several times. Since the purification of enzymes usually is vary time and
cost
intensive, an immobilization and reuse of the enzyme contributes to a
considerable
reduction of the costs. Another advantage is the high degree of purity of the
reaction
products which inter alia is due to the fact that the reaction conditions can
be better
controlled when immobilized enzymes are used. The insoluble linear glucans
yielded
as reaction products can then be easily purified further.
There are many carrier materials available for the immobilization of proteins
which
can be coupled to the carrier material either by covalent or non-covalent
links (for an
overview see: Methods in Enzymology Vol. 135, 136 and 137). Widely used
carrier
materials are, e.g., agarose, cellulose, polyacryiamide, silica or nylon.
A further possibility of the use of proteins having amylosucrase activity is
to use them
for the production of cyclodextrins. Cyclodextrins are produced by the
degradation of
starch by the enzyme cyclodextrin transglycosylase (EC 2.4.1.19) which is
obtained
from the bGctsrium Bacillus macerans. Due to the branching of starch only
about
40% of the glucose units can be converted to cyclodextrins using this system.
By
providing substantially pure proteins having amylosucrase activity it is
possible to
synthesize cyclodextrins on the basis of sucrose under the simultaneaus action
of
amylosucrase and cyclodextrin transglycosylase, with the amylosucrase
catalyzing
the synthesis of linear glucans from sucrose and the cyciodextrin
transglycosylase
catalyzing the conversion of these glucans into cyclodextrins.
Abbreviations used
IPTG isopropyl f3-D-thiogalacto-pyranoside
Media and solutions used
YT medium 8 g bacto-tryptone
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1~
g yeast extract
5 g NaCI
ad 1000 ml with ddH20
YT plates YT medium with 15 g bacto-agar/
1000 ml
Lugol's solution 12 g KI
6912
ad 1.8 I with ddH20
The examples serve to illustrate the invention.
Example 1
Isolation of a genomic DNA sequence coding for an amylosucrase activity from
Neisseria polysaccharea
For the isolation of a DNA sequence coding for an amylosuc~ ase activity from
Neisseria polysaccharea first a genomic DNA library was established. Neisseria
polysaccharea cells were cultured on "Columbia blood agar" (Difco) for 2 days
at
37°C. The resulting colonies were harvested from the plates. Genomic
DNA was
isolated according to the method of Ausubel et al. (in: Current Protocols in
Molecular
Biology (1987), ,l. Wiley & Sons, NY) and processed. The DNA thus obtained was
partially digested with the restriction endonuclease Sau3A. The resulting DNA
fragments were ligated into the 8amHl digested vector pBluescript SK(-). The
ligation
products were transformed in E. coli XL1-Blue cells. For their selection, the
cells were
plated onto YT plates with ampicillin as selection marker. The selection
medium
additionally contained 5% sucrose and 1 mM IPTG. After incubation over night
at
37°C the bacterial colonies that had formed were stained with iodine by
placing
crystalline iodine into the lid of a petri dish and placing the culture dishes
with the
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16
bacteria colonies for 10 min each conversely onto the petri dish. The iodine
which
evaporated at room temperature stained some regions of the culture dishes that
contained amylose-like glucans blue. From bacteria colonies that showed a blue
corona plasmid DNA was isolated according to the method of Birnboim & Doly
(Nucleic Acids Res. 7 (1979), 1513-1523). Said DNA was retransformed in E.
coil
SURE cells. The transformed cells were plated onto YT plates with ampicillin
as
selection marker. Positive clones were isolated.
Example 2
Sequence analysis of the genomic DNA insert of the plasmid pNB2
From an E. coil clone obtained according to working example 1 a recombinant
plasmid was isolated. Restriction analyses showed that said plasmid was a
ligation
product consisting of two vector molecules and an approx. 4.2 kb long genomic
fragment. The plasmid was digested with the restriction endonuclease Pstl and
the
genomic fragment was isolated (GeneClean, Bio101). The fragment thus obtained
was ligated into a pBluescript II SK vector linearized with Pstl, resulting in
a
duplication of the Psti and Smal restriction sites. The ligation product was
transformed in E. coli cells and the latter were plated on ampicillin plates
for
selection. Positive clones were isolated. From one of these clones a plasmid
was
isolated and part of the sequence of its genomic DNA insert was determined by
standard techniques using the dideoxy method (Sanger et al., Proc. Natl. Acad.
Sci.
USA 74 (1977), 5463-5467). The entire insert is approx. 4.2 kbp long. The
nucleotide
sequence was determined and is indicated in SEQ ID NO. 1.
Example 3
Expression of an extracellular amylosucrase activity in transformed E. coli
cells
(a) Detection of an amylosucrase activity during growth on YT plates
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For the expression of an extracellular amylosucrase activity, E. coli cells
were
transformed with the isolated plasmid vector according to standard techniques.
A
colony of the transformed strain was incubated on YT plates (1.5% agar; 100
Ng/ml
ampicillin; 5% sucrose; 0.2 mM IPTG) over night at 37°C. The
amylosucrase activity
was detected by subjecting the colonies to iodine vapor. Amylosucrase-
expressing
colonies exhibit a blue corona. Amylosucrase activity can be observed even if
no
IPTG was present, probably due to the activity of the endogenous amylosucrase
promoters.
(b) Detection of an amylosucrase activity during growth in YT medium
For the expression of an extracellular amylosucrase activity, E. coil were
transformed
with the isolated plasmid vector according to standard techniques. YT medium
(100
pg/ml ampicillin; 5% sucrose) was inoculated with a colony of the transfarmed
strain.
The cells were incubated over night at 37°C under constant agitation
(rotation mixer;
150-200 rpm). The products of the reaction catalyzed by amylosucrase were
detected by adding Lugol's solution to the culture supernatant, leading to
blue
staining.
(c) Detection of the amylosucrase activity in the culture supernatants of
transformed E. coil cells which were cultivated without sucrose
For the expression of an extracellular amylosucrase activity, E. coil cells
were
transformed with the isolated plasmid vector according to standard techniques.
YT
medium (100 pglml ampicillin) was inoculated with a colony of the transformed
strain.
The cells were incubated over night at 37°C under constant agitation
(rotation mixer;
150-200 rpm). Then the cells were removed by centrifugation (30 min,
4°C, 5500
rpm, JA10 Beckmann rotor). The supernatant was filtered through a 0.2 pm
filter
(Schleicher & Schuell) under sterile conditions.
Detection of an amylosucrase activity was carried out by
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~s
(i) incubating the supernatant on a sucrose-containing agar plate.. 40 pl of
the
supernatant were placed in a whole punched into an agar plate (5% sucrose in
50 mM sodium citrate buffer pH 6.5) and incubated at least for one hour at
37°C. The products of the reaction catalyzed by amylosucrase were
detected
by staining with iodine vapor. Presence of the reaction products produces a
blue stain.
(ii) or by gel electrophoretic separation of the proteins of the supernatant
in a
native gel and detection of the reaction products in the gel after incubation
with
sucrose. 40-80N1 of the supernatant were separated by gel electrophoresis on
an 8°/° native polyacrylamide gel (0.375 M Tris pH 8.8) at a
voltage of 100 V.
The gel was then twice equilibrated 15 min with approx. 100 ml 50 mM sodium
citrate buffer (pH 6.5) and incubated over night at 37°C in sodium
citrate buffer
pH 6.5/5% sucrose. In order to make the reaction product of the reaction
catalyzed by amylosucrase visible, the gel was rinsed with Lugol's solution.
Bands having amylosucrase activity were stained blue.
Example 4
In vitro production of glucans with partially purified amylasucrase
For the expression of an extracellular amylosucrase activity, E. coli cells
were
transformed with the isolated plasmid vector according to standard techniques.
YT
medium (100 Ng/ml ampicillin) was inoculated with a colony of the transformed
strain.
The cells were incubated over night at 37°C under constant agitation
(rotation mixer;
150-200 rpm). Then the cells were removed by centrifugation (30 min,
4°C, 5500
rpm, JA10 Beckmann rotor). The supernatant was filtered through a 0.2 Nm
filter
(Schleicher 8~ Schuell) under' sterile conditions.
The supernatant was then concentrated by 200 times using an Amicon chamber
(YM30 membrane having an exclusion size of 30 kDa, company Arnicon) under
pressure (p=3 bar). The concentrated supernatant was added to 50 ml of a
sucrose
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l9
solution (5% sucrose in 50 rnM sodium citrate buffer pH 6.5). The entire
solution was
incubated at 37°C. Whitish insoluble polysaccharides are formed.
Example 5
Characterization of the reaction products synthesized by amylosucrase from
Example
4
The insoluble reaction products described in Example 4 are soluble in 1 M
NaOH.
The reaction products were> characterized by measuring the absorption maximum.
Approx. 100 mg of the isolated reaction products (wet weight) were dissolved
in 200
NI 1 M NaOH and diluted with H20 1:10. 900 NI of 0.1 M NaOH and 1 ml Lugol's
solution were added to 100 pl of this dilution. The absorption spectrum was
measured between 400 and 700 nm. The maximum is 605 nm (absorption maximum
of amylose: approx. 614 nm).
HPL.C analysis of the reaction mixture of Example 4 on a CARBOPAC PA1 column
(DIONEX) showed that in addition to the insoluble products soluble products
were
also formed. These soluble products are short-chained polysaccharides. The
chain
length was between approx. 5 and approx. 60 glucose units. To a smaller
extent,
however, even shorter or longer molecules could be detected.
With the available analytical methods it was not possible to detect branching
in the
synthesis products.
Example 6
Expression of an intracellular amylosucrase activity in transformed E. coli
cells
Using a polymerase chain reaction (PCR) a fragment was amplified from the
isolated
plasmid vector which com,orises the nucleotides 981 to 2871 ;,' the sequence
depicted in SEQ fD NO. 1. The following oligonucleotides were used as primers:
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TPN2 5' - CTC ACC ATG GGC ATC TTG GAC ATC - 3'
(SEQ ID N(~. 3)
TPC1 5' - CTG CCA TGG TTC AGA CGG CAT TTG G - 3'
(SEQ ID N(~. 4)
The resulting fragment contains the coding region for amylosucrase except for
the
nucleotides coding for the '16 N-terminal amino acids. These amino acids
comprise
the sequences that appear to be necessary for the secretion of the enzyme from
the
cell. Furthermore, this PCR fragment contains 88 by of the 3' untranslated
region. By
way of the primers used Nc;ol restriction sites were introduced into both ends
of the
fragment.
After digestion with the restriction endonuclease Ncol the resulting fragment
was
ligated with the Ncol digested expression vector pMex 7. The ligation products
were
transformed in E. coli cells and transformed clones were selected. Positive
clones
were incubated over night at 37°C on YT plates (1.5% agar; 100 Nglml
ampiciflin; 5%
sucrose; 0.2 mM IPTG). After subjecting the plates to iodine vapor no blue
staining
could be observed in the area surrounding the bacteria colonies, but the
intracellular
production of glycogen could be detected (brown staining of transformed cells
in
contrast to no staining in nontransformed XL1-Blue cells). In order to examine
the
functionality of the protein, transformed cells cultivated on YT medium were
broken
up by ultrasound and the obtained crude extract was pipetted onto sucrose-
containing agar plates. After subjecting the plates to iodine vapor a blue
stain could
be observed.
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1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: PlantTec Biotechnologie GmbH Forschung &
Entwicklung
(B) STREET: Hermannswerder 14
(C) CITY: Potsdam
(E) COUNTRY: Germany
(F) POSTAL CODE (ZIP): 14473
(ii) TITLE OF INVENTION: Nucleic acid molecules encoding an
amylosucrase
(iii) NUMBER OF SEQUENCES: 4
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release X1.0, Version X1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4173 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISMz Neisseria polysaccharea
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1971..3878
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GATCGCCTTC GCCCAATTGC GACCAAAGTT TTTTGGTAAA CAGCTTGGGG TTGTTCTCGA 60
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2
TGACTTTGTTGGCGATTTTGAGAATCCGCGCGGTGGAGCGGTAGTTTTGCTCCAGTTTGA120
TGACCTTCATCTGCGGATAGTTTTCCTGCATTTTGCGCAGGTTTTCCATGTTCGCGCCGC180
GCCATGCGTAGATGGACTGGTCGTCGTCGCCGACGGCGGTAAACATCCCTTCCGCGCCGG240
TCAGAAGTTTCATCAACGTAAA7.'TGGCAGGTATTCGTATCTTGGCATTCGTCAACCAGCA300
GATAACGCAGCCGCCGCTGCCA7.'TTGTTGCGCACTTCGCTGTTTTGCTGCAACAGCACGG360
CAGGCAGGCGGATTAAGTCATCGAAGTCCACTGCCTGATAGCTTTGTAAGGTTTCCTGAT420
AGCTCGCATACACGCGTGCGGTTTGTTGTTCCCAAATGTTCGATGCCGTCTGAACGACAT480
CTTCAGGCGTTTTTAAATCGTTTTTCCAAAGGGAAATTTGAT~TTGCGCTTTGAATGTGG540
CTTCTTTGCCCGTACCGCCTAAGAGTTCGCCGATGATTTTCGCGCTGTCGGTAGAGTCGA600
GGATGGAGAAGTTTTTTTTGTAACCGATATGGTTCGCCTCTTCGCGCAAAATCT7'CATGC660
CCAAAGAATGGAACGTGCAAATTGTCAGCCCGCGCGTTTGCGATTTGGGCAGCATTTTGG720
CGACGCGCTCCTGCATTTCCGCP,GCGGCTTTGTTGGTAAAGGTAATCGCGGCGACGGTAT780
GCGGCAGATAGCCGACATTGACFATCAAATGCTTGATTTTTTGAGTAATCACGCCGGTTT840
TTCCGCTGCCCGCGCCTGCAAGGACGAGCAGGGGGCCGCCGAGGTAGCGGACGGCTTCGA900
GCTGTTGGGGATTGAGTTTCA"L'CATGTTTTGATGCCGTCTGAAATCAGTCTGCGCCGCTT960
TCGAGGCAGTCGAGTGCCGCA~~GGAGGGCGGATACGCCGATTTGCCCCGGCGCGGAGTTT1020
TGCGTTCCCGAACCGAACGTGATGCTTGAGCCGAACACCTGTCCGGCAAGGCGGCTGACC1080
GCCCCCTTTTGCCCCATCGACATCGTAACAATCGGTTTGGTGGCAAGCTCTTTCGCTTTG1140
AGCGTGGCAGAAAGCAAAGTCAGCACGTCTTCCGCGCTTTGCGGCATCACCGCAATTTTG1200
CAGATGTCCGCGCCGCAGTCC'rCCATCTGTTTCAGACGGCATACGATTTCTTCTTGCGGC1260
GGCGTGCGGTGAAACTCATGA'rTGCAGAGCAGGGCGGCGATGCCGTTTTTTTGAGCATGC1320
GCCACGGCGCGCCGGACGGCGGTTTCGCCGGAAAAAAGCTCGATATCGATAATGTCGGGC1380
AGGCGGCTTTCAATCAGCGAG'_CGAGCAGTTCAAAATAATAATCGTCCGAACACGGGAAC1440
GAGCCGCCTTCGCCATGCCGTC_'TGAACGTAAACAGCAGCGGCTTGTCGGGCAGCGCGTCG1500
CGGACGGTCTGCGTGTGGCGC:'~ATACTTCGCCGATGCTGCCCGCGCATTCCAAAA.AATCG1560
GCGCGGAACTCGACGATATCG.-'~AGGGCAGGTTTTTGATTTGGTCAAGTACGGCGGAAAGT1620
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3
ACGGCGGCATCGCGGGCGAC GCGATTTTGGTGCGTCCGCTTCCGATAACG 1680
AAGCGGCACG
GTGTTTTTGACGGTCAGGCTGGTGTGCATGGCGGTTGTTGCG3CTGAAAGGAACGGTAAA 1740
GACGCAATTATAGCAAAGGCACAGGCAATGTTTCAGACGGCATTTCTGTGCGGCCGGCTT 1800
GATATGAATCAAGCAGCATCCGCATATCGGAATGCAGACTTGGCACAAGCCCTGTCTTTT 1860
CTAGTCAGTCCGCAGTTCTTGCAGTATGATTGCACGACACGCCCTACACGGCATTTGCAG 1920
GATACGGCGGCAGACCGCCGGTC(3GAAACTTCAGAATCGGAGCAGGCATCATG TTG 1975
Met Leu
1
ACC CCC ACG CAG CAA GTC G(3T TTG ATT TTA CAG TAC CTC AAA ACA CGC 2024
Thr Pro Thr Gln Gln Val G.L~,r Leu Ile Leu Gln Tyr Leu Lys Thr Arg
10 L5
ATC TTG GAC ATC TAC ACG CCC GAA CAG CGC GCC GGC ATC GAA AAA TCC 2072
Ile Leu Asp Ile Tyr Thr Pro Glu Gln Arg Ala Gly Ile Glu Lys Ser
20 25 30
GAA GAC TGG CGG CAG TTT TCG CGC CGC ATG GAT ACG CAT TTC CCC AAA 2120
Glu Asp Trp Arg Gln Phe Se r Arg Arg Met Asp Thr His Phe Pro I,ys
35 40 45 50
CTG ATG AAC GAA CTC GAC AGC GTG TAC GGC AAC AAC GAA GCC CTG CTG 2168
Leu Met Asn Glu Leu Asp Se r Val Tyr GIy Asn Asn Glu Ala Leu Leu
55 60 65
CCT ATG CTC- GAA ATG CTG CTG GCG CAG GCA TGG CAA AGC TAT TCC CAA 2216
Pro Met Leu Glu Met Leu Leu Ala Gln Ala Trp G1n Ser Tyr Ser Gln
70 75 80
CGC AAC TCA TCC TTA AAA GAT ATC GAT ATC GCG CGC GAA AAC AAC CCC 2264
Arg Asn Ser Ser Leu Lys Asp Ile Asp Ile Ala Arg Glu Asn Asn Pro
85 90 95
GAT TGG ATT TTG TCC AAC AAA CAA GTC GGC GGC GTG TGC TAC GTT GAT 2312
Asp Trp Ile Leu Ser Asn Lys Gln Val Gly Gly Val Cys Tyr Val Asp
100 105 110
TTG TTT GCC GGC GAT TTG AAG GGC TTG AAA GAT AAA ATT CCT TAT TTT 2360
Leu Phe Ala Gly Asp Leu Lyres Gly Leu Lys Asp Lys Ile Pro Tyr Phe
115 120 125 130
CAA GAG CTT GGT TTG ACT TAT CTG CAC CTG ATG CCG CTG TTT AAA TGC 2408
Gln Glu Leu Gly Leu Thr Tyr Leu His Leu Met Pro Leu Phe Lys Cys
135 140 145
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4
CCT GAA GGC AAA AGC GAC GGC GGC TAT GCG GTC AGC AGC TAC CGC GAT 2456
Pro Glu Gly Lys Ser Asp Gly Gly Tyr Ala Val Ser Ser Tyr Arg Asp
150 155 160
GTC AAT CCG GCA CTG GGC A.CA ATA GGC GAC TTG CGC GAA GTC ATT GCT 2504
Val Asn Pro Ala Leu Gly Thr Ile Gly Asp Leu Arg Glu Val Ile Ala
165 170 175
GCG CTG CAC GAA GCC GGC ATT TCC GCC GTC GTC GAT TTT ATC TTC AAC 2552
Ala Leu His G1u Ala Gly Ile Ser Ala Val Val Asp Phe Ile Phe Asn
180 185 190
CAC ACC TCC AAC GAA CAC GAA TGG GCG CAA CGC TG~ GCC GCC GGC GAC 2600
His Thr Ser Asn Glu His Glu Trp Ala Gln Arg Cys Ala Ala Gly Asp
195 200 205 2I0
CCG CTT TTC GAC AAT TTC TAC TAT ATT TTC CCC GAC CGC CGG ATG CCC 2648
Pro Leu Phe Asp Asn Phe Tyr Tyr Ile Phe Pro Asp Arg Arg Met Pro
215 220 225
GAC CAA TAC GAC CGC ACC CTG CGC GAA ATC TTC CCC GAC CAG CAC CCG 2696
Asp Gln Tyr Asp Arg Thr Leu Arg Glu Ile Phe Pro Asp Gln His Pro
230 235 240
GGC GGC TTC TCG CAA CTG GAA GAC GGA CGC TGG GTG TGG ACG ACC TTC 2744
Gly Gly Phe Ser Gln Leu Glu Asp Gly Arg Trp Val Trp Thr Thr Phe
245 250 255
AAT TCC TTC CAA TGG GAC TTG AAT TAC AGC AAC CCG TGG GTA TTC CGC 2792
Asn Ser Phe Gln Trp Asp Leu Asn Tyr Ser Asn Pro Trp Val Phe Arg
260 265 270
GCA ATG GCG GGC GAA ATG CTG TTC CTT GCC AAC TTG GGC GTT GAC ATC 2840
Ala Met Ala G1y Glu Met Leu Phe Leu Ala Asn Leu Gly Val Asp Ile
275 280 285 290
CTG CGT ATG GAT GCG GTT GCC TTT ATT TGG AAA CAA ATG GGG ACA AGC 2888
Leu Arg Met Asp Ala Val Ala Phe Ile Trp Lys Gln Met Gly Thr Ser
295 300 305
TGC GAA AAC CTG CCG CAG GCG CAC GCC CTC ATC CGC GCG TTC AAT GCC 2936
Cys Glu Asn Leu Pro Gln A1a EIis Ala Leu Ile Arg Ala Phe Asn Ala
310 315 320
GTT ATG CGT ATT GCC GCG CC~ GCC GTG TTC TTC AAA TCC GAA GCC ATC 2984
Val Met Arg Ile Ala Ala Pro Ala Val Phe Phe Lys Ser Glu Ala Ile
325 330 335
GTC CAC CCC GAC CAA GTC G'TC CAA TAC ATC GGG CAG GAC GAA TGC CAA 3032
Val His Pro Asp Gln Val Val Gln Tyr Il.e Gly Gln Asp Glu Cys Gln
340 345 350
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ATC GGT TAC AAC CCC CTG CAA ATG GCA TTG TTG TGi AAC ACC CTT GCC 3080
Ile Gly Tyr Asn Pro Leu Gln Met Ala Leu Leu Trp Asn Thr Leu Ala
355 360 365 370
ACG CGC GAA GTC AAC CTG CTC CAT CAG GCG CTG ACC TAC CGC CAC AAC 3128
Thr Arg Glu Val Asn Leu Leu His Gln Ala Leu Thr Tyr Arg His Asn
375 380 385
CTG CCC GAG CAT ACC GCC.''CGG GTC AAC TAC GTC CGC AGC CAC GAC GAC 3176
Leu Pro Glu His Thr Ala 'Crp Val Asn Tyr Val Arg Ser His Asp Asp
390 395 400
ATC GGC TGG ACG TTT GCC GAT GAA GAC GCG GCA TAT CTG GGC ATA AGC 3224
Ile Gly Trp Thr Phe Ala Asp Glu Asp Ala Ala Tyr Leu Gly Ile Ser
405 410 415
GGC TAC GAC CAC CGC CAA ':CTC CTC AAC CGC TTC TTC GTC AAC CGT TTC 3272
Gly Tyr Asp His Arg Gln I?he Leu Asn Arg Phe Phe Val Asn Arg Phe
420 42 5 430
GAC GGC AGC TTC GCT CGT GGC GTA CCG TTC CAA TAC AAC CCA AGC ACA 3320
Asp Gly Ser Phe Ala Arg Gly Val Pro Phe Gln Tyr Asn Pro Ser 7',~r
435 440 445 450
GGC GAC TGC CGT GTC AGT GCGT ACA GCC GCG GCA TTG GTC GGC TTG GCG 3368
Gly Asp Cys Arg Val Ser Gly Thr Ala Ala Ala Leu Val Gly Leu Ala
455 460 465
CAA GAC GAT CCC CAC GCC C;TT GAC CGC ATC AAA CTC TTG TAC AGC ATT 3416
Gln Asp Asp Pro His Ala Val Asp Arg Ile Lys Leu Leu Tyr Ser Ile
470 475 480
GCT TTG AGT ACC GGC GGT C:TG CCG CTG A'rT TAC CTA GGC GAC GAA GTG 3464
Ala Leu Ser Thr Gly Gly Leu Pro Leu Ile Tyr Leu Gly Asp Glu Val
485 490 495
GGT ACG CTC AAT GAC GAC GAC TGG TCG CAA GAC AGC AAT AAG AGC GAC 3512
Gly Thr Leu Asn Asp Asp Asp Trp Ser Gln Asp Ser Asn Lys Ser Asp
500 505 510
GAC AGC CGT TGG GCG CAC C:GT CCG CGC TAC AAC GAA GCC CTG TAC GCG 3560
Asp Ser Arg Trp Ala His Arg Pro Arg Tyr Asn Glu Ala Leu Tyr Ala
515 520 525 530
CAA CGC AAC GAT CCG TCG ACC GCA GCC GGG CAA ATC TAT CAG GGC TTG 3608
Gln Arg Asn Asp Pro Ser 'Thr Ala Ala Gly Gln Ila Tyr Gln Gly Leu
535 540 545
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CGC CAT ATG ATT GCC GTC C:GC CAA AGC AAT CCG CGC TTC GAC GGC GGC 3656
Arg His Met Ile Ala Val Arg Gln Ser Asn Pro Arg Phe Asp Gly Gly
550 555 560
AGG CTG GTT ACA TTC AAC ACC AAC AAC AAG CAC ATC ATC GGC TAC ATC 3704
Arg Leu Val Thr Phe Asn Thr Asn Asn Lys His Ile Ile Gly Tyr Ile
565 570 575
CGC AAC AAT GCG CTT TTG CJCA TTC GGT AAC TTC AGC GAA TAT CCG CAA 3752
Arg Asn Asn Ala Leu Leu Al.a Phe Gly Asn Phe Ser G.lu Tyr Pro ,~ln
580 '_;85 590
ACC GTT ACC GCG CAT ACC C'.TG CAA GCC ATG CCC TTC AAG GCG CAC GAC 3800
Thr Val Thr Ala His Thr he:u Gln Ala Met Pro Phe Lys Ala His Asp
595 600 605 610
CTC ATC GGT GGC AAA ACT C~TC AGC CTG AAT CAG GAT TTG ACG CTT CAG 3848
Leu Ile Gly GIy Lys Thr Val Ser Leu Asn Gln Asp Leu Thr Leu Gln
6I5 620 625
CCC TAT CAG GTC ATG TGG C:TC GAA ATC GCC TGACGCACGC TTCCCAAATG 3898
Pro Tyr Gln Val Met Trp heu Glu Ile A1a
630 635
CCGTCTGAAC CGTTTCAGAC GGCATTTGCG CCGAAGCGGA CGGTAGTCCC CAAAAGGGAA 3958
ACATGCGATA ATAGCCGCCC ATC.'ACATCCC GCGCCGCAGC CCGTGTTGCG CCGCATCCCA 4018
CATACCGCAT TTGTTCCGGA GTt~ACCCCAA TGTCAGACGA CAAAAGCAAA GCCCTTGCCG 4078
CCGCACTGGC GCAAATCGAA AAAAGTTTCG GCAAAGGCGC CATCATGAAA ATGGACGGCA 4138
GCCAGCAGGA AGAAAACCTC GAF.GTCATTT CCACC 4173
(2) INFORMATION FOR SEQ :ID NO: 2:
(i) SEQUENCE CHA_~~.CTERISTICS:
(A) LENGTH: 636: amino acids
(B} TYPE: amino acid
(D) TOPOLOGY: .l.i.near
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Leu Thr Pro Thr Gln C:ln Va1 Gly Leu Ile Leu Gln Tyr Leu Lys
1 5 10 15
Thr Arg Ile Leu Asp Ile Tyr Thr Pro G1u Gln Arg Ala Gly Ile Glu
20 25 30
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Lys Ser Glu Asp Trp Arg Gln Phe Ser Arg Arg Met Asp Thr His Phe
35 40 45
Pro Lys Leu Met Asn G1u heu Asp Ser Val Tyr Gly Asn Asn Glu Ala
50 'i5 60
Leu Leu Pro Met Leu Glu Met Leu Leu Ala Gln Ala Trp Gln Ser Tyr
65 70 75 80
Ser Gln Arg Asn Ser Ser heu Lys Asp Ile Asp Ile Ala Arg Glu Asn
85 90 95
Asn Pro Asp Trp Ile Leu Ser Asn Lys G1n Val Gly Gly Val Cys Tyr
100 105 110
Val Asp Leu Phe Ala Gly Asp Leu Lys Gly Leu Lys Asp Lys Ile Pro
115 120 125
Tyr Phe Gln Glu Leu Gly heu Thr Tyr Leu His Leu Met Pro Leu Phe
130 _L35 140
Lys Cys Pro Glu Gly Lys Ser Asp Gly Gly Tyr Ala Val Ser Ser Tyr
145 150 155 160
Arg Asp Val Asn Pro Ala heu Gly Thr Ile Gly Asp Leu Arg Glu Val
165 170 175
Ile Ala Ala Leu His Glu Ala Gly Ile Ser Ala Val Val Asp Phe Ile
180 185 190
Phe Asn His Thr Ser Asn Glu His Glu Trp Ala Gln Arg Cys Ala Ala
195 200 205
Gly Asp Pro Leu Phe Asp Asn Phe Tyr Tyr Ile Phe Pro Asp Arg Arg
210 :?:.5 220
Met Pro Asp Gln Tyr Asp Arg Thr Leu Arg Glu Ile Phe Pro Asp Gln
225 230 235 240
His Pro Gly Gly Phe Ser Gln Leu Glu Asp G1y Arg Trp Val Trp Thr
245 250 255
Thr Phe Asn Ser Phe Gln 'Crp Asp Leu Asn Tyr Ser Asn Pro Trp Val
260 265 270
Phe Arg Ala Met Ala Gly Glu Met Leu Phe Leu Ala Asn Leu Gly Val
275 280 285
Asp Ile Leu Arg Met Asp Ala Val Ala Phe Ile Trp Lys Gln Met Gly
290 295 300
CA 02342124 2001-03-O1
WO 00/14249 8 PC's /EP98/05573
Thr Ser Cys Glu Asn Leu Pro Gln Ala His Ala Leu Ile Arg Ala Phe
305 310 315 320
Asn Ala Val Met Arg Ile >~la Ala Pro Ala Val Phe Phe Lys Ser Glu
325 330 335
Ala Ile Val His Pro Asp Glln Val Val Gln Tyr Ile Gly Gln Asp Glu
340 345 350
Cys Gln Ile Gly Tyr Asn Pro Leu Gln Met Ala Leu Leu Trp Asn Thr
355 360 365
Leu Ala Thr Arg Glu Val Asn Leu Leu His Gln Ala Leu Thr Tyr Arg
370 375 380
His Asn Leu Pro Glu His T'hr Ala Trp Val Asn Tyr Val Arg Ser His
385 390 395 400
Asp Asp Ile Gly Trp Thr F~he Ala Asp Glu Asp Ala Ala Tyr Leu Gly
405 410 415
Ile Ser G1y Tyr Asp His Arg Gln Phe Leu Asn Arg Phe Phe Val Asn
420 425 430
Arg Phe Asp Gly Ser Phe P.la Arg Gly Val Pro Phe Gln Tyr Asn Pro
435 440 445
Ser Thr Gly Asp Cys Arg V'al Ser Gly Thr Ala Ala Ala Leu Val Gly
450 455 460
Leu Ala Gln Asp Asp Pro H:is Ala Val Asp Arg Ile Lys Leu Leu Tyr
465 470 475 480
Ser Ile Ala Leu Ser Thr Gly Gly Leu Pro Leu Ile Tyr Leu Gly Asp
485 490 495
Glu Val Gly Thr Leu Asn A.sp Asp Asp Trp Ser Gln Asp Ser Asn Lys
500 505 510
Ser Asp Asp Ser Arg Trp A.la His Arg Pro Arg Tyr Asn Glu Ala Leu
515 520 525
Tyr Ala Gln Arg Asn Asp Pro Ser Thr Ala Ala Gly Gln Ile Tyr Gln
530 535 540
Gly Leu Arg His Met Ile A.la Val Arg G1n Ser Asn Pro Arg Phe Asp
545 550 555 560
Gly Gly Arg Leu Val Thr Phe Asn Thr Asn Asn Lys His Ile Ile Gly
565 570 575
CA 02342124 2001-03-O1
WO 00/14249 ~ PCT/EP98/05573
Tyr Ile Arg Asn Asn Ala Leu Leu Ala Phe Gly Asn Phe Ser Glu Tyr
580 585 590
Pro Gln Thr Val Thr Ala :EIis Thr Leu Gln Ala Met Pro Phe Lys Ala
595 600 605
His Asp Leu Ile Gly Gly :Lys Thr Val Ser Leu Asn Gln Asp Leu Thr
610 6:15 620
Leu Gln Pro Tyr G1n Val. Mc~t Trp Leu Glu Ile Ala
625 630 635
(2) INFORMATION FOR SEQ :LD NO: 3:
(i) SEQUENCE CHARAC'CERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: :_inear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTTOPd: /desc = "oligonucleotide"
(iii) HYPOTHETICAL: NO
(iv) AP:T'_'-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Dleisseria polysaccharea
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CTCACCATGG GCATCTTGGA CATC 24
(2) INFORMATION FOR SEQ I:D NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nuc.Leic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION': /desc = "oligonucleotide"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NC
CA 02342124 2001-03-O1
WO 00/14249 1 ~ PC'T/EP98/05573
(vi) ORIGINAL SOURCE:
(A) ORGANISM: lJeisseria polysaccharea
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CTGCCATGGT TCAGACGGCA TT'CGG 25
