Note: Descriptions are shown in the official language in which they were submitted.
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RECOMBINANT ~-GALACTOSIDASE ENZYME
5Statement of Government Interest
This invention was made with government support
under NMRDC Grant Number N00014-90-J-1638. As such, the
government has certain rights in the invention.
FIELD OF THE I~v~llON
This invention relates to a recombinant enzyme for
use in the removal of type B antigens from the surface of
cells in blood products, thereby converting type B blood
products to type O blood products and type AB blood products
to type A blood products without otherwise affecting the
structure and function of the cells in the blood products.
This invention further relates to methods of cloning and
expressing said recombinant enzyme. More particularly, this
invention is directed to a recombinant coffee bean
~-galactosidase enzyme, a recombinant vector which encodes
coffee bean ~-galactosidase, methods of cloning and
expressing said recombinant ~-galactosidase enzyme, the use
of said recombinant ~-galactosidase enzyme to cleave
galactose sugar residues, most particularly ~1,3-linked
galactose residues, which are responsible for blood group B
specificity, and a method of removing type B antigens from
the surface of cells in type B and AB blood products using
said recombinant coffee bean ~-galactosidase enzyme by
contacting said enzyme with blood products so as to remove
the terminal moiety of the B-antigenic determ;nAnt from the
surface of cells (for example, erythrocytes~ in said blood
products. The recombinant coffee bean ~-galactosidase
enzyme of this invention provides a readily available and
cost-efficient enzyme which can be used in the removal of
type B antigens from the surface of cells in type B and AB
blood products. Treatment of type B blood products with the
recombinant enzyme of this invention provides a source of
cells free of the B antigen, which blood products are
-
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thereby rendered useful in transfusion therapy in the same
manner as O type blood products.
BACKGROUND OF THE INVENTION
As used herein, the term "blood products" includes
whole blood and cellular components derived from blood,
including erythrocytes (red blood cells) and platelets.
There are more than thirty blood group (or type)
systems, one of the most important of which is the ABO
system. This system is based on the presence or absence of
antigens A and/or B. These antigens are found on the
surface of erythrocytes and on the surface of all
endothelial and most epithelial cells as well. The major
blood product used for transfusion is erythrocytes, which
are red blood cells containing hemoglobin, the principal
function of which is the transport of oxygen. Blood of
group A contains antigen A on its erythrocytes. Similarly,
blood of group B contains antigen B on its erythrocytes.
Blood of group AB contains both antigens, and blood of group
0 contains neither antigen, but does contain a structure
known as H antigen.
The blood group structures are glycoproteins or
glycolipids and considerable work has been done to identify
the specific structures making up the A and B determinants
or antigens. It has been found that the blood group
specificity is determined by the nature and linkage of
monosaccharides at the ends of the carbohydrate chains. The
carbohydrate chains are attached to a peptide or lipid
backbone which is embedded in the lipid bi-layer of the
membrane of the cells. The most important (immuno-dominant
or immuno-determinant) sugar has been found to be
N-acetylgalactosamine for the type A antigen and galactose
for the type B antigen.
Blood of group A contains antibodies to antigen B.
Conversely, blood of group B contains antibodies to antigen
A. Blood of group AB has neither antibody, and blood group
O has both. A person whose blood contains either (or both)
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of the anti-A or anti-B antibodies cannot receive a
transfusion of blood cont~;n;ng the corresponding
incompatible antigen(s). If a person receives a transfusion
of blood of an incompatible group, the blood transfusion
5 recipient's antibodies coat the red blood cells of the
transfused incompatible group and cause the transfused red
blood cells to agglutinate, or stick together. Transfusion
reactions and/or hemolysis (the destruction of red blood
cells) may result therefrom.
In order to avoid red blood cell agglutination,
transfusion reactions and hemolysis, transfusion blood type
is cross-matched against the blood type of the transfusion
recipient. For example, a blood type A recipient can be
safely transfused with type A blood which contains
compatible antigens. Because type 0 blood contains no A or
B antigens, it can be transfused into any recipient with any
blood type, i.e., recipients with blood types A, B, AB or O.
Thus, type 0 blood is considered "universal", and may be
used for all transfusions. Hence, it is desirable for blood
banks to maintain large quantities of type O blood.
However, there is a paucity of blood type O donors.
Therefore, it is useful to convert types A, B and AB blood
to type O blood in order to maintain large quantities of
universal blood products.
In an attempt to increase the supply of type O
blood, methods have been developed for converting certain
type A, B and AB blood to type O blood (containing only the
H antigen structure). For example, U.S. Patent No.
4,330,619 entitled "Enzymatic Conversion of Red Cells for
Transfusion" issued May 18, 1982 to Goldstein ("the '619
Patent"), which is incorporated herein by reference, is
directed to a process for converting type B erythrocytes to
the H antigen type (or type O) and type AB erythrocytes to
type A utilizing a!-galactosidase enzyme. The process for
converting B and AB erythrocytes which is described in the
'619 Patent includes the steps of equilibrating B or AB
erythrocytes, contacting the equilibrated erythrocytes and
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purified ~-galactosidase for a period of time sufficient to
convert the B antigen in the erythrocytes to the H antigeln,
removing the ~-galactosidase enzyme from the erythrocytes
and re-equilibrating the erythrocytes. U.S. Patent No.
4,427,777 entitled "Enzymatic Conversion of Red Cells for
Transfusion" issued January 24, 1984 to Goldstein, which
Patent is incorporated herein by reference, is directed to
compositions free of B antigens wherein B antigens are
removed from the compositions utilizing ~-galactosidase and
the process described in the '61~ Patent.
a-galactosidase enzymes are characterized (and
thereby named) by their ability to cleave a-linked galactose
sugar groups. In isolating or identifying these enzymes,
their activity is assessed in the laboratory by evaluating
cleavage of synthetic substrates which mimic the sugar
groups cleaved by the enzymes, with p-nitrophenyl
~-D-galactopyranoside derivatives of the target sugar groups
being commonly used. Synthetic substrates are useful in
enzyme identification and isolation procedures (the
quantitative cleavage of these synthetic substrates can be
used to readily distinguish (and thereby identify) enzymes
isolated from different sources). However, these synthetic
substrates and other oligosaccharide substrates are
structurally simple and small-sized, and mimic only a
portion of the natural glycoproteins and glycolipid
structures (glycoconjugates) which are of primary concern,
those being the B antigens on the surface of cells.
~ -galactosidase enzymes from a number of sources
have been purified, sequenced, cloned and expressed. (See,
for example, Fellinger et al., Yeast, Vol. 7, pp. 463-473
(1991) (expression of guar a-galactosidase); Yagi et al~,
Archives Biochem. and Biophysics, Vol. 280, pp. 61-67 (1990)
(purification of Ehrlich ascites tumor cells and fluid
~-galactosidase); Bahl et al., Meth. EnzYmol., Vol. XXVIII,
pp. 728-743 (1972) (purification of AsPerqillus niger
~-galactosidase); and Courtois et al., Meth. EnzYmol.,
Vol. VIII, pp. 565-571 (1966) (purification of coffee bean
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~-galactosidase)). However, not all ~-galactosidase enzymes
are appropriate for use in removing B antigens from the
surface of cells in blood products.
In determining whether an enzyme is appropriate
for use in removing B antigens from the surface of cells,
one must consider the following enzyme characteristics:
substrate specificity, specific activity or velocity of the
substrate cleavage reaction, and pH optimum. Substrate
specificity is measured in the Km value, which measures the
binding constant or affinity of an enzyme for a particular
substrate. The lower a Km value, the more tightly an enzyme
binds its substrate. The velocity of an enzyme cleavage
reaction is measured in the Vmax, the reaction rate at a
saturating concentration of substrate. A higher Vmax
indicates a faster cleavage rate. The ratio of these two
parameters, Vmax/Km, is a measure of the overall efficiency
of an enzyme in reacting with (cleaving) a given substrate.
A higher Vmax/Km indicates greater enzyme efficiency. For
successful and clinically applicable removal of B antigens
from the surface of cells, the enzyme must be sufficiently
active at or above a pH at which the cells being treated
that can be maintained, that being pH 5.6 (or above) for red
cells. Therefore, the pH optimum and activity profile of an
appropriate enzyme must still provide reasonable enzyme
activity at this pH.
The pH optimum of Ehrlich cell ~-galactosidase
enzyme centers near 4.5, irrespective of substrate (see Yagi
et al., Archives Biochem. and Biophysics, Vol. 280, pp.
61-67 (1990)). The pH optimum or Ehrlich cell
~-galactosidase has been found to be 4.5 for water-soluble
fluorogenic substrates and oligosaccharides (see Dean et
al., J. Biol. Chem., Vol. 254, pp. 10006-10010 (1979)). In
contrast, the pH optimum of coffee bean ~-galactosidase for
the fluorogenic substrate PNP-~-Gal is 6.0, indicating that
the coffee bean enzyme exhibits significant activity at or
above a pH at which cells are treated for removal of B
antigens.
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Coffee bean ~-galactosidase enzyme shows a Vmax/km
value of 236 at pH 6.0 toward PNP-~-gal, whereas
~-galactosidases isolated from human cells (see Dean and
Sweeley, J. Biol. Chem., Vol. 254, pp. 10006-10010 (1979))
or Ehrlich ascites tumor (see Yagi et al., Archives Biochem.
and Biophysics, Vol. 280, pp. 61-67 (1990)) only have a
Vmax/Km value of between 7.59 and 9.9 at pH 4.5-4.6 (using
4-Me-~-gal or PNP-~-gal). Furthermore, in the comparison of
substrate specificity of ~-galactosidases from coffee bean
and Ehrlich ascites tumor cells, Yagi et al. found that
coffee bean ~-galactosidases showed much higher Vmax/km
values toward oligosaccharide substrates such as raffinose
(450 fold), gal~1,3 gal (180 fold) and galactor~nn~n (30
fold). Based on this study, they concluded that coffee bean
~-galactosidase showed a relatively broad substrate
specificity, suggesting that it is suited for cleaving many
kinds of terminal ~-galactosyl linkages. Of all the
~-galactosidases studied, the one obtained from coffee bean
demonstrates the highest activity in removing terminal
~1,3-linked galactose residues from glycoconjugates. This
makes the coffee bean enzyme a most appropriate enzyme in
the study and performance of enzymatic blood conversion.
Prior to the present invention, it was necessary
to purify the ~-galactosidase enzyme from a coffee bean
source, a process which is time consuming and can be
expensive. Because coffee bean ~-galactosidase can be used
to convert B and AB blood products, a need has arisen to
develop a coffee bean ~-galactosidase enzyme source which is
more readily available. In addition, a need has arisen to
develop a coffee bean ~-galactosidase enzyme useful in type
B and AB blood product conversion, the production of which
enzyme is cost-efficient. Further, it is desirable to
provide a more readily available and controlled source of
enzyme, that source being a cloned and expressed enzyme,
which would provide an enzyme source which is more
consistent and which is readily purified at less cost.
Additionally, a recombinant, cloned enzyme would allow for
-
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specific protein sequence modifications, which can be
introduced in order to generate an enzyme with further
optimized specific activity, substrate specificity and pH
range.
It is therefore an object of this invention to
provide recombinant coffee bean ~-galactosidase enzyme for
use in the removal of B antigens from the surface of cells
in blood products.
It is another object of this invention to provide
recombinant coffee bean a-galactosidase enzyme for use in
the removal of B antigens from the surface of cells in blood
products wherein said enzyme is readily available and may be
manufactured on a cost-efficient basis.
It is a further object of this invention to
provide methods of cloning and expressing recombinant coffee
bean ~-galactosidase enzyme useful in the removal of B
antigens from the surface of cells in blood products.
It is another object of this invention to provide
a recombinant vector containing a nucleotide sequence
encoding coffee bean ~-galactosidase enzyme useful for
expressing recombinant coffee bean ~-galactosidase enzyme or
for modifying said enzyme through recombinant methods.
It is a still further object of this invention to
provide a recombinant coffee bean ~-galactosidase enzyme
which is capable of removing terminal ~1,3-linked galactose
residues from substrates and glycoconjugates.
It is yet another object of this invention to
provide a method of removing B antigens from the surface of
cells in blood products using recombinant coffee bean
~-galactosidase enzyme.
SU~ RY OF THE INrVENTION
This invention is directed to a recombinant coffee
bean ~-galactosidase enzyme capable of cleaving ~1,3-linked
glycoside linkages on cells. This invention is further
directed to a recombinant vector containing a nucleotide
sequence encoding coffee bean ~-galactosidase.
_ _
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Additionally, this invention is directed to a method of
producing coffee bean ~-galactosidase, and to a method of
removing B antigens from the surface of cells which method
comprises contacting cells with recombinant coffee bean
~-galactosidase enzyme for a period of time sufficient to
remove the B antigens from the surface of the cells.
BRIEF DESCRIPTION OF THE DRAWINGS
The above brief description, as well as further
objects and features of the present invention, will be more
fully understood by reference to the following detailed
description of the presently preferred, albeit illustrative/
embodiments of the present invention when taken in
conjunction with the accompanying drawings wherein:
Figure 1 represents the nucleotide and deduced
amino acid sequence of full-length cDNA encoding coffee bean
~-galactosidase;
Figure 2 represents a comparison of sequence
homology of ~-galactosidase from coffee bean, guar
(CYamoPsis tetraqonoloba), human placenta, yeast
(Saccharomyces cerevisiae) and fungi (Aspergillus niaer) as
aligned using the computer program PROSIS and manual
arrangement;
Figure 3 represents immunoprecipitation with
polyclonal antibody of cloned coffee bean ~-galactosidase
expressed in vitro in rabbit reticulocyte lysate and wheat
germ extract, as analyzed by SDS-PAGE and autoradiographed;
and
Figure 4 represents Western blot analysis of
recombinant coffee bean ~-galactosidase expressed in
transfected sf9 insect cells using antibody against purified
coffee bean ~-galactosidase.
Figure 5 represents the sequence of the plasmid
p~F-BZ around the 5' cloning site (EcoRI) of the insert.
The two arrows indicate the signal cleavage sites as
indicated by N-terminal sequencing of the secreted enzyme.
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Figure 6 represents the chromatogram from the
cation exchange chromatography purification of the
recombinant ~-galactosidase enzyme produced by the Pichia
pastoris expression system.
Figure 7 represents the SDS-PAGE analysis of the
chromatography purified recombinant a-galactosidase enzyme
produced by the Pichia pastoris expression system. Lane 1,
supernatant of Pichia pastoris culture; lane 2, unbound
fraction from the column; lane 3 r fraction #55; lane 4,
fraction #65; lane 5, fraction #80; lane 6, fraction #150;
lane 7, native ~-galactosidase enzyme; and lane 8, size
markers (kDa).
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to a recombinant coffee
bean ~-galactosidase enzyme capable of cleaving ~1,3-linked
glycoside linkages on cells. The recombinant coffee bean
~-galactosidase enzyme of the invention has a molecular
weight of about 42 kDa, and has about 80% amino acid
sequence homology with guar a-galactosidase enzyme. This
invention is further directed to a recombinant vector
containing a nucleotide sequence which encodes coffee bean
~-galactosidase.
In addition, this invention is directed to a
method of producing coffee bean ~-galactosidase, and to a
method of removing B antigens from the surface of cells
which method comprises contacting cells with a recombinant
coffee bean ~-galactosidase enzyme for a period of time
sufficient to remove the B antigens from the surface of the
cells.
Group B erythrocytes may be treated with
~ ~-galactosidase isolated from coffee beans to cleave the
terminal ~1,3-linked galactose residues responsible for
~ blood group B specificity in order to convert the group B
erythrocytes serologically to group O erythrocytes. Hence,
it is desirable to have readily available purified coffee
bean ~-galactosidase. In order to produce purified
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--10--
~-galactosidase enzyme in large quantities and improve its
enzymatic properties, the inventors have isolated the cDNA
clone for coffee bean ~-galactosidase.
As discussed hereinabove, ~-galactosidase has been
purified from several sources. However, only coffee bean
~-galactosidase cleaves ~1,3-linked galactose residues
responsible for blood group B specificity. Hence, only
coffee bean ~-galactosidase can be used to convert type B
blood products to type O blood products, and type AB blood
products to type A blood products.
The full length cDNA which encodes coffee bean
~-galactosidase is represented in SEQ ID NO:1 and Figure 1~
A DNA vector containing a sequence encoding coffee bean
~-galactosidase was deposited under the Budapest Treaty with
the American Type Culture Collection, Rockville, Maryland,
on September 8, 1993, tested and found viable on September
14, 1993, and catalogued as ATCC #75556.
Methods which are well known to those skilled in
the art can be used to construct expression vectors
containing the coffee bean ~-galactosidase coding sequence,
with appropriate transcriptional/translational signals for
expression of the enzyme in the corresponding expression
systems. Appropriate org~n; ~r~, cell types and expression
systems include: cell-free systems such as a rabbit
reticulocyte lysate system, prokaryotic bacteria, such as E.
coli, eukaryotic cells, such as yeast, insect cells,
mammalian cells (including human hepatocytes or Chinese
hamster ovary (CH0) cells), plant cells or systems, and
animal systems including oocytes and transgenic animals.
The entire coffee bean ~-galactosidase coding
sequence or functional fragments of functional equivalents
thereof may be used to construct the above expression
vectors for production of functionally active enzyme in the
corresponding expression system. Due to the degeneracy of
the DNA code, it is anticipated that other DNA sequences
which encode substantially the same amino acid sequence may
be used. Additionally, changes to the DNA coding sequence
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which alter the amino acid sequence of the coffee bean
~-galactosidase enzyme may be introduced which result in the
expression of functionally active enzyme. In particular,
amino acid substitutions may be introduced which are based
on similarity to the replaced amino acids, particularly with
regard to the charge, polarity, hydrophobicity,
hydrophilicity, and size of the side chains of the amino
acids.
Once a recombinant coffee bean ~-galactosidase
enzyme is cloned and expressed, said enzyme can be used to
remove B antigens from the surface of cells in blood
products. Type B antigens can be removed from the surface
of erythrocytes by contacting the erythrocytes with the
recombinant coffee bean ~-galactosidase enzyme of the
invention for a period of time sufficient to remove the B
antigens from the surface of the erythrocytes.
ExamPle
In order to assess the relative abilities of
~-galactosidase enzyme isolated from different sources to
remove el,3-linked galactose residues from red cells, l00 ml
of type B red blood cells were treated with isolated
~-galactosidases from yeast (S. cerevisiae), fungi (A.
niger), guar (C. tetraqonoloba) and coffee bean~ The
treatment conditions and digestion results are provided
below in Table I, below. Digestion of terminal sugars
(~1,3-linked galactose residues) was determined by assessing
reduction or elimination of the agglutination of treated
cells in the presence of polyclonal anti-B antibody. No
detectable change in agglutination indicated no digestion~
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TABLE I
Source of Digestion
~-Galactosidase Conditions Results
Yeast 90 U/ml RBC No Digestion
26~C, 17 hours
pH 5.6
10 Fungi 1600 U/ml RBC No Digestion
26~C, 6 hours
pH 5.6
Guar 20 U/ml RBC* No Digestion
26~C, 2.25 hours
pH 5.6
Coffee Bean 90 U/ml RBC Complete
26~C, 90 minutes Digestion
pH 5.6
*Digestion with coffee bean enzyme under the same conditions
removes at least 95% of B antigens.
None of the a-galactosidases isolated from sources
other than coffee bean showed any significant activity in
removing the terminal a-linked galactose residues from the
red blood cell surfaces. In contrast, coffee bean
a-galactosidase demonstrated high activity in removing
terminal al,3-linked galactose residues from glycoconjugates
on the red cell surfaces. Hence, coffee bean
~-galactosidase is appropriate for use in enzymatic blood
group B type conversion.
Peptide Sequencing of
a-Galactosidase Purified from Coffee Beans
In order to sequence the coffee bean
a-galactosidase, coffee bean a-galactosidase was purified to
apparent homogeneity from green coffee beans. The procedure
used for purification of a-galactosidase from coffee beans
was developed and optimized in the laboratory to provide
pure a-galactosidase enzyme (demonstrating a single band on
SDS polyacrylamide gel electrophoresis) with optimal yields
under large scale conditions. This provided sufficient
- =
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-13-
material to be used for treatment of type B blood products
to remove B antigens on a clinical scale. The following
procedure was utilized: Green Santos beans were frozen in
either liquid nitrogen or in a -70~C freezer to facilitate
grinding in a Waring blender. The crude powder obtained was
~ homogenized with H2O (at a ratio of 4 liters H2O per kg
ground beans) and allowed to stand overnight at room
temperature. The following day repeated homogenization was
necessary to achieve a rich first extract, which was then
expressed (under pressure) through multiple layers of
cheesecloth. The homogenate was subjected to a second
extraction as described above. The pH of the combined
extracts was adjusted to 4.0 with acetic acid and then 0.5%
diatomaceous earth (Super Cell filter aid, available from
Cellulo Company, a division of Gosmer Enterprises, Cranford,
NJ) was added. The formed heavy precipitate was removed
with a filter funnel. The filtrate was then processed
through the chromatography steps as indicated in Table II.
After steps 1, 2, 4 and 5 in Table II, the
collected main fraction was concentrated and then
equilibrated for the next step. This was accomplished with
a Pellicon Cassette System using a 10,000 MW cutoff
membrane. PCS buffer pH 5.6 had the following composition:
58 mM dibasic sodium phosphate, 21 mM citric acid, 77 mM
sodium chloride. Concentrate phosphate-citrate buffer was
prepared by titrating 50 mM citric acid with 100 mM dibasic
sodium phosphate pH 3.7. Sepharose divinylsulfone galactose
was prepared according to the procedure described by Ersson
et al., Biochem. et Biophys. Acta, Vol. 494, pp. 51-60
(1977), which is incorporated herein by reference. PBE94
(Polybuffer exchanger 4) is available from Pharmacia, Inc~
Other similar anion exchange resins, which are well known in
the art and available commercially, can be used in place of
DE53 in the above procedure, particularly other DEAE
(diethylaminoethyl) resins.
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-14-
...
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_r ~ o = ~ q; = _ -- = O al = t_~
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CA 02211417 1997-07-2~
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-15-
Alternative methods for purification of
~-galactosidase from coffee beans have been reported by
Haibach et al., Biochem. et Biophys. Res. Comm., Vol. 181,
No. 3, pp. 1564-1571 (1991) and Harpaz et al., Biochem et
Biophys. Acta, Vol. 341, pp. 213-221 (1974), however, these
purification procedures have been found to provide lower
overall yields of enzyme from coffee beans on sufficient
scale for clinical use than that described above. The
enzyme activity of coffee bean ~-galactosidase was assayed
according to the procedure described by Kuo et al., Enzyme
Microb. Technol., Vol. 5, pp. 285-290 (1983)), which is
incorporated herein by reference, using a final
concentration of PNP-a-Gal of 1.25 mM.
Microsequencing of the unblocked mature enzyme
provided an amino-terminal sequence (N-pep) of 19 residues~
In order to obtain additional peptide sequences including
internal sequences, 0.2 mg of the purified ~-galactosidase
was treated with 2 mg of cyanogen bromide in 70~ formic acid
for 24 hours at room temperature in the dark. The peptides
were isolated by reverse phase HPLC. Two peptide sequences,
2-pep and 3-pep, were then determined by automated gas phase
microsequencing. The sequences of the three peptides
(N-pep, 2-pep and 3-pep) are indicated in Figure 1.
Figure 1 represents the full length cDNA encoding
coffee bean ~-galactosidase. Three peptide sequences,
N-pep, 2-pep and 3-pep, which were obtained from purified
coffee bean ~-galactosidase, are underlined below the
deduced amino acid sequence. The first 15 amino acids
comprise a putative signal peptide which is cleaved during
biosynthesis. Therefore, the mature coffee bean
~-galactosidase enzyme is comprised of the amino acids
16-378 of Figure 1. The potential N-linked glycosylation
site is double-underlined at amino acid residues 160-162.
The polyadenylation signal (AA'Tl~AA) at the position
ntl361-1366 is boxed.
The oligonucleotides, CB1, CB4 through CB9, are
shown with arrows to indicate 5' to 3' direction. Based on
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-16-
the sequence of the peptide, N-pep, the CBl* was designed as
5'ACA(CT)CCA(T)CCA(T)ATGGNTGGAA. Accordingly, the CB4*,
based on the sequence of the peptide, 3-pep, has the
sequence 5'-TGT(A)GGT(GA)GTNAGG(CA)ACG(A)TACAT. CB1 bears
the least codon degeneracies in the peptide sequence of
N-pep as determined by the computer program "Primer"
(Scientific and Educational Software, Inc.). Because CNBr
cleaves at the C-terminal side of the methionine residue,
the codon for methionine was included at the 3' end of
oligonucleotide CB4. Because there have not been any
reported genes cloned from the coffee plant, the preference
codons used in the designing of the oligonucleotides were
chosen based on those of other plants listed in the codon
usage table.
Molecular Cloning of the Full Length
cDNA Encoding Coffee Bean ~-Galactosidase
In order to perform molecular cloning of the full
length cDNA encoding coffee bean ~-galactosidase, total RNA
was prepared from 2 grams of dried green coffee beans by
using the Extract-A-plant RNA Isolation kit (ClonTech)
according to the manufacturer's procedure. The quality of
the isolated RNA was confirmed by denaturing agarose gel
electrophoresis. The messenger RNA was purified from the
total RNA by using an oligo-dT column (ClonTech).
In order to isolate the specific cDNA encoding the
~-galactosidase, cDNA using isolated coffee bean mRNA was
prepared according to the stAn~rd procedure known in the
art for reverse transcription. A mixture of oligo dT and
random primer was used as the reverse transcription primer
in the reaction to avoid the 3' bias when oligo-dT is used
alone. The cDNA then provided the template in a PCR
reaction for 35 cycles, 94~C 1 minute, 50~C 2 minutes and
72~C 3 minutes. In the presence of oligonucleotides CB1 and
CB4, this PCR procedure produced a fragment of approximately
l.lkb. This fragment, designated BZ, was cloned directly
into the pCRII vector (Invitrogen) for further analysis.
The sequencing data indicated that BZ was highly homologous
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with guar ~-galactosidase. (BZ corresponds to sequence
nucleotides (nt) 168 to 1234 in Figure 1). Furthermore, its
deduced amino acid sequence matched the peptide sequences
obtained from purified coffee bean ~-galactosidase,
providing evidence for its authenticity as coffee bean
~-galactosidase cDNA.
In order to obtain the full-length cDNA coding for
the coffee bean ~-galactosidase, the technique of 5' and 3'
RACE was applied (see Frohman et al., Proc. Natl. Acad. Sci.
USA, Vol. 85, pp. 8998-9002 (1988)). For 5' RACE, the
oligonucleotide, CB8, was first used as a primer for the
reverse transcription from coffee bean mRNA. The following
procedure was then followed for 5' RACE as in the kit
manufacturer's protocol (Bethesda Research Laboratories
(BRL)). The second oligonucleotide, CB6, together with the
universal primer was used to amplify the 5' region upstream
of the coding sequence by PCR. Since no distinctive DNA
band was visible on the agarose gel after two PCR
amplifications, the PCR product mixture was cloned into the
pCRII vector and screened by hybridizing the colonies with
the radioactively labeled l.lkb fragment BZ (sequence
ntl68-1234 in Figure 1). The positive colonies were picked
for plasmid preparation. The sequencing of the plasmid
indicated that the DNA fragment, designated S'BZ, obtained
by the 5' RACE technique contained a 240bp overlap (the
sequence between CB1 and CB6) with the BZ, and about a 170bp
further upstream sequence which includes the N-terminus of
the mature enzyme and the putative signal peptide sequence.
To isolate the 3' sequence, downstream from the
fragment BZ, the cDNA was reverse-transcribed from coffee
bean mRNA by using a primer, PI, which has the sequence
5'-GACTCGAGTCGACATCGA-(T)17. PCR amplification was then
carried out with a specific primer CB7 (sequence nt940-957
in Figure 1) and an adapter primer, PII. PII has the same
sequence as PI except that it lacks seventeen thymidine
residues at its 3' end. The PCR product was analyzed on 1~
low melting point agarose gel and a distinctive band of
CA 02211417 1997-07-2~
W O 96/23869 PCTfUS96/01212
-18-
about 500 bp long was visualized. The fragment designated
3' BZ, was isolated and cloned into the pCRII vector for
sequencing. The sequence data indicated that the 5' region
of the fragment, 3'BZ, is identical to the 3' region
(sequence from nt940-1234) of the BZ. An in-frame stop
codon TGA was localized at ntl236-1238, confirming that the
peptide sequence, 3-pep, obtained from the purified coffee
bean ~-galactosidase represents the C-terminal sequence of
the protein.
The three DNA fragments, 5'BZ, BZ and 3'BZ, were
linked together by PCR to reconstitute the full length clone
for coffee bean ~-galactosidase. Oligonucleotide CB9 was
made, which corresponded to sequence nt77-94 shown in Figure
1. cDNA was synthesized from coffee bean mRNA using the 3'
RACE technique as previously described. A 1.35kb fragment
was amplified by PCR using two primers CB9 and PII, and was
then cloned into the pCR II vector. The plasmids thus
generated contained the 1.35kb insert in both orientations.
The plasmid pCR-BZ6 has the insert downstream of an SP6
promoter, which was used in in vitro expression. A second
plasmid pCR-BZ7 containing the opposite insert orientation
was used for subcloning the ~-galactosidase cDNA into a
baculovirus expression vector. The sequence of the 1.35kb
product matched with the corresponding sequences from the
three separate fragments, 5'BZ, BZ and 3'BZ, confirming the
authentic sequence of the coffee bean ~-galactosidase cDNA
shown in Figure 1.
Characterization of the
Coffee Bean ~-Galactosidase cDNA Clone
Next, the coffee bean ~-galactosidase cDNA clone
was characterized. The sequence shown in the Figure 1
encodes a protein having a molecular weight of 42kDa, which
closely approximates the size of the purified coffee bean
~-galactosidase as estimated on SDS-PAGE. Three peptide
sequences, N-pep, 2-pep and 3-pep, which were derived from
purified enzyme, are underlined in Figure 1. These
sequences matched the deduced amino acid sequences. This
CA 02211417 1997-07-2~
WO 96/23869 PCT/US96/01212
--19--
confirms that the cDNA clone isolated from coffee bean RNA
encodes ~-galactosidase. Since the mature protein starts at
leucine, amino acid residue 16 in Figure 1, the first 15
residues may serve as a signal sequence which is removed
5 following translation. There is an in-frame stop codon
(TGA) at position nt66-68, suggesting that the first
downstream ATG at position ntlO2-104 is the initiation codon
for in vivo synthesis of ~-galactosidase precursor. The
sequence at residues 160-162 (boxed) is the only possible
10 site for N-glycosylation. However, purified coffee bean
c~-galactosidase does not bind to ConA Sepharose, indicating
that there is no or minimal glycosylation of c~-galactosidase
enzyme synthesized in coffee beans.
The first plant ~-galactosidase cDNA was cloned
15 from guar (see Overbeeke et al., Plant Molecular Biology,
Vol. 13, pp. 541-550 (1989)). Guar ~-galactosidase encodes
a protein of 411 residues haviny a molecular weight of
45 kDa. Although both ~-galactosidases from guar and coffee
bean show comparable activities toward the synthetic
20 substrate PNP-~-gal, their specificities toward
oligosaccharide chains are very different. Guar
~-galactosidase primarily cleaves a 1,6 glycoside linkages,
whereas coffee bean ~-galactosidase cleaves a! 1,3 and 1,4
linkages. Thus, the guar ~-galactosidase is unable to
25 cleave significant amounts of terminal ~1,3-linked galactose
residues from the cell surface of B group red cells. Figure
2 represents the sequence homology of ~-galactosidase from
different sources. The aminc acid sequences of
~-galactosidase from coffee bean (coffee), CyamoPsis
30 tetraqonoloba (guar), human placenta (human), Saccharomyces
cerevisiae (yeast) and Asperqillus niger (Aspergillus) were
aligned by using the computer program PROSIS (Hitachi
Software Engineering Corp., Ltd.) and manual arrangement.
The gaps are created in order to show maximum similarity.
35 The numbers above the sequences indicate the relative
position of each amino acid sequence. The sequences of
yeast and AsPerqillus niqer ~-galactosidases are truncated
CA 02211417 1997-07-2~
W 096/23869 PCTAUS96/01212
-20-
at the C-terminus, (indicated by *), removing 38 and 103
residues respectively. Identical or conservatively
substituted amino acid residues (five out of six or more at
the same position) are boxed according to the equivalent
amino acid list. 1: A,S,T,P and G; 2: N,D,E and Q; 3: H,R
and K; 4: M,L,I and V; and 5: F,Y and W.
By using the protein analysis program PROSIS
(Hitachi Software Engineering Corp., Ltd.~, it was
determined that the deduced amino acid sequences of the
coffee bean cDNA clone and the guar a-galactosidase cDNA
clone share approximately 80% overall homology, even though
their signal peptide sequences bear little similarity. The
coffee bean a-galactosidase also shows amino acid sequence
homology with a-galactosidase from human (59%), yeast (58%)
and Asperqillus niqer (52%).
As shown in Figure 2, when the deduced amino acid
sequences of a-galactosidase from these five different
sources are aligned, many residues are well conserved in all
of these a-galactosidases. However, these sequences share
little, if any, homology with a-galactosidase isolated from
E. coli (see Liljestrom et al., Nucleic Acids Res., Vol. 15,
pp~ 2213-2220 (1987)). Recently, two more ~-galactosidase
cDNAs have been reported from Klebsiella Pneumoniae (see
Hama et al., J. Biol. Chem., Vol. 267, pp. 18371-18376
(1992)) and a Streptococcus mutant (see Aduse-Opoku et al.,
J. Gen. Microbiol., Vol. 137, pp. 2271-2272 (1991)).
However, they do not bear any sequence homology with known
a-galactosidases at the amino acid level.
In Vitro ExPression of Clone Coffee Bean a-Galactosidase
In order to perform in vitro expression of cloned
coffee bean ~-galactosidase in a transcription-translation
coupled system (TNT system, Promega), two plasmids were
used: pCR-BZ6, which contains a-galactosidase cDNA
downstream from the SP6 promoter, and the vector pCRII as a
control. The protein(s) were expressed in both rabbit
reticulocyte lysate and wheat germ extract and then
CA 022ll4l7 l997-07-2~
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immunoprecipitated by the polyclonal antibody raised against
purified coffee bean ~-galactosidase. All the samples were
analyzed by a SDS-PAGE and autoradiographed as shown in
Figure 3. Figure 3 represents in vitro expression and
immunoprecipitation of cloned coffee bean ~-galactosidase.
Approximately 2 mg of plasmids (pCR and pCR-BZ6) were added
to 50 ml mixture of TNT rabbit reticulocyte lysate in the
presence of 35S-methionine and SP6 DNA polymerase according
to the Promega recommended protocol. After incubation at
30~C for 90 minutes, the samples (5 ml of each reaction)
were loaded onto a 12~ gel SDS-PAGE (lanes 2 and 3).
Immunoprecipitation was carried out by incubating the same
expression mixtures (20 ml) with antisera (1 ml) raised
against purified coffee bean ~-galactosidase. The
immunoprecipitated samples were analyzed in lanes 4 and 5.
Lanes ~ through 9 show the results of the same experiments
except that TNT wheat germ extract was used instead of
rabbit reticulocyte lysate. The arrow at right indicates
the expressed ~-galactosidase. The molecular weight
standard (lane 1) is shown at left.
When the rabbit reticulocyte lysate was used,
multiple proteins (molecular weights ranging from 25 kDa to
42 kDa) were expressed from ~-galactosidase cDNA and
recognized by the antibody (lanes 3 and 5). In contrast,
only one predominant band was visualized on the gel (lanes
7 and 9) by using wheat germ extract. This band, as
indicated by an arrow, migrated as a protein of approximate
molecular weight 42 kDa, the predicted size based on the
a-galactosidase cDNA sequence. The apparent discrepancy of
the translation patterns in these two extracts may possibly
be due to the fact that since the ~-galactosidase cDNA was
isolated from coffee bean, its expression may be more
optimized in the wheat germ extract. The multiple bands
observed in the rabbit reticulocyte lysate might result from
alternative initiation or premature termination. The
results show that the 42 kDa protein expressed in vitro is
coffee bean ~-galactosidase.
CA 022ll4l7 l997-07-2~
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-22-
Functional Expression of
Coffee Bean a-Galactosidase in Insect Cells
Coffee bean ~-galactosidase was then functionally
expressed in insect cells. Many eukaryotic proteins have
been expressed in insect cells infected with recombinant
baculovirus (see King et al., The Baculovirus Expression
System: A Laboratory Guide, Chapman ~ Hall, New York
(1992)). Since insect cells carry out the
post-translational processing events that occur in
eukaryotic cells, including glycosylation and signal peptide
cleavage, proteins produced by such baculovirus expression
systems are similar to their natural counterparts in
biological activity, structure and antigenicity.
Coffee bean ~-galactosidase cDNA was subcloned
from the plasmid pCR-BZ7 into the unique NotI/BamHI sites of
a baculovirus expression vector pVL 1392 (PharMingen),
generating the plasmid pVL-BZ. Expression of
~-galactosidase cDNA was thus under the control of a strong
viral promoter (polyhedrin promoter). The plasmid pVL-BZ
was co-transfected into sf9 insect cells with baculoGold DNA
(PharMingen), a lethal deletion of the virus DNA, according
to the procedure suggested by the manufacturer. Through
homologous recombination with a complementary sequence in
the plasmid, viable virus cont~;n;ng the ~-galactosidase
cDNA was thus reconstituted inside the insect cells and
released into the medium. The transfection supernatant
(1 ml) was then added to fresh sf9 cells (2 x 106). After
incubation at 27~C for three days, the supernatant was
harvested and used for virus amplification one more time in
order to obtain a high titer of virus.
In order to detect the expression of
~-galactosidase in the transfected sf9 cells, a Western blot
was carried out by using antibody against purified coffee
bean ~-galactosidase. Figure 4 represents expression of
recombinant coffee bean ~-galactosidase in insect cells
(sf9). The supernatants and cells were collected after the
second amplification and analyzed by Western blot with
polyclonal antibody against purified coffee bean
-
CA 02211417 1997-07-2~
W096/23869 PCT~S96101212
-23-
~-galactosidase. The supernatant and cells from pVL-BZ
transfection are shown in lanes l and 2, respectively. The
supernatant (lane 3) and cells (lane 4) from wild-type virus
transfection were used as a negative control. Lane 5 is
~-galactosidase purified from coffee bean. Molecular weight
standards are listed at left.
As shown in Figure 4, the coffee bean
~-galactosidase was expressed in the sf9 cells transfected
with the plasmid pVL-BZ (lane 2) but not in cells
transfected with wild-type virus (lane 4). Its migration on
the gel was similar to that of purified enzyme (lane 5). In
addition, secretion of the expressed protein in the culture
supernatant (lane l) was not detected.
The ~-galactosidase activity was tested by
directly incubating pVL-BZ transfected sf9 cells with
l.25 lluvi PNP-a-gai (pH 6.5j. During the incubation, the
PNP-~-galactosidase substrate enters the cells by diffusion.
After incubation at 37~C for an hour the reaction was
stopped by adding l ml of borate buffer (pH 9.8) and
absorbance at 405 nm was measured. The total proteins in
the reaction were precipitated by addition of
trichloroacetate and measured by Bio-Rad Protein Assay
(Bio-Rad). The average activity of ~-galactosidase
expressed in the insect cells was approximately 300 uni~s,
one unit being defined as l nmol of substrate hydrolyzed per
hour at 37~C. The endogenous activity in the wild-type
virus infected cells was undetectable under such conditions.
The recombinant coffee bean ~-galactosidase enzyme
of the invention can be used to remove terminal galactose
residues from the non-reducing end of carbohydrate chains
(from polysaccharides and glycoconjugates), particularly
those galactose residues which are ~l,3-linked.
The recombinant coffee bean ~-galactosidase enzyme
of the invention can be used to convert type B erythrocytes
to type O and type AB erythrocytes to type A. Specifically,
the recombinant coffee bean ~-galactosidase enzyme of the
invention is put into contact with cells having B
CA 022ll4l7 l997-07-2~
W 096/23869 PCTrUS96/01212
antigenicity for a period of time sufficient to remove the
B antigens from the surface of the cells. More
specifically, erythrocytes having B antigenicity are washed
and then equilibrated in isotonic phosphate-citrate-sodium
5 chloride (PCS) at pH 5.5 and 5.6, sequentially,
c~-galactosidase is added at a concentration of from 75 to
200 U/ml, and the mixture is incubated at 26~C. More
preferably, the ~-galactosidase is added at a concentration
of 200 U/ml and the mixture is incubated at 26~C for 2.25
10 hours. Most preferably, the final hematocrit of the
erythrocytes is 65 to 75g~. In addition, cells transformed
with a recombinant vector which encodes coffee bean
~x-galactosidase can be cultured and coffee bean
~-galactosidase can be recovered from the culture, which
15 coffee bean ~-galactosidase can then be used to remove B
antigens from the surface of cells and blood products.
Expression of Coffee Bean ~-Galactosidase in Pichia pastoris
Coffee bean ~-galactosidase cDNA was subcloned in
20 the EcoRI site of Pichia pastoris expression vector pPIC9
(Invitrogen Corp., San Diego, CA) generating the plasmid
p~F-BZ with the sequence around the 5' cloning site of the
insert as shown in Figure 5. The expression of ~x-
galactosidase was under the control of the methanol
25 inducable promoter AOX1. The expressed protein was secreted
into the culture media via the signal sequence of yeast ~
mating factor. After transformation of Pichia pastoris, one
transformant with the highest ~-galactosidase activity was
selected for large-scale production in a fermentor.
30 Approximately eleven copies of the ~-galactosidase was
incorporated into the Pichia pastoris genome based upon a
dot blot experiment. The level of ~-galactosidase secreted
into the Pichia pastoris culture media reached 400 mg/L of
culture. After removal of the Pichia pastoris cells from
35 the fermentation culture, the recombinant ~-galactosidase in
the supernatant was purified by cation exchange
chromatography. The chromatogram from the purification is
-
CA 02211417 1997-07-2~
W 096/23869 PCTrUS96/01212
-25-
presented in Figure 6. The chromatography purified enzyme
was then subjected to SDS-PAGE analysis.
N-terminal sequencing of the purified protein
~ suggested that the signal sequence was cleaved at two
positions as indicated by the two arrows in Figure 5,
generating two forms of ~-galactosidase with Phe or Leu as
the N-terminal residue in approximately equal molarity.
Characterization of the native coffee bean ~-galactosidase
and the recombinant coffee bean ~-galactosidase indicated
that despite the mixture of N-terminal amino acid residues,
the recombinant ~-galactosidase exhibited essentially the
same molecular size, specific activity, pH optima and
kinetic parameters as the native enzyme.
Although the invention herein has been described
with reference to particular embodiments, it is to be
understood that these embodiments are merely illustrative of
various aspects of the invention~ Thus, it is to be
understood that numerous modifications may be made in the
illustrative embodiments and other arrangements may be
devised without departing from the spirit and scope of the
invention.
CA 02211417 1997-07-25
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-26-
SEOUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: ALEX ZHU AND JACK GOLDSTEIN
(ii) TITLE OF INVENTION: RECOMBINANT
ALPHA-GALACTOSIDASE ENZYME AND CDNA ENCODING SAID
ENZYME
(iii~ NUMBER OF SEQUENCES: 11
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: AMSTER, ROTHSTEIN & EBENSTEIN
(B) STREET: 90 PARK AVENUE
(C) CITY: NEW YORK
(D) STATE: NEW YORK
(E) COUNTRY: U.S.A.
(F) ZIP: 10016
(v) COh~U'l'~K READABLE FORM:
(A) MEDIUM TYPE: 3. 5 INCH 1. 44 Mb STORAGE
DI~K~
(B) COh~U'1'~;K: IBM PC COMPATIBLE
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: ASCII
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: NOT YET ASSIGNED
(B) FILING DATE: NOT YET ASSIGNED
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: CRAIG J. ARNOLD
(B) REGISTRATION NUMBER: 34,287
(C) REFERENCE/DOCKET NUMBER: 63475/71
(iX) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 697-5995
(B) TELEFAX: (212) 286-0854 Or 286-0082
(C) TELEX: TWX 710-581-4766
(2) INFORMATION FOR SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1409
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: DOUBLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE:
(A) DESCRIPTION: OLIGONUCLEOTIDE
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
CA 02211417 1997-07-2~
W096/23869 PCT/US96/01212
--27--
(vi ) ORIGINAL SOURCE:
(A) ORGANISM: GREEN COFFEE BEAN
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 1
CT AGT AAA AAA AAG CCA CCC AAA AGC TGG TGC TCC GAG CTT CGT TAT 47
TGA TGC TTT TAT GTT TCT TGA CGG TTG AAA AAC GTT GGT GCT TCC GCT 95
CGC CGG ATG GTG AAG TCT CCA GGA ACC GAG GAT TAC ACT CGC AGG AGC 143
Met Val Lys Ser Pro Gly Thr Glu Asp Tyr Thr Arg Arg Ser
CTT TTA GCA AAT GGG CTT GGT CTA ACA CCT CCG ATG GGG TGG AAC AGC 191
Leu Leu Ala Asn Gly Leu Gly Leu Thr Pro Pro Met Gly Trp Asn Ser
TGG AAT CAT TTC CGT TGT AAT CTT GAT GAG AAA TTG ATC AGG GAA ACA 239
Trp Asn His Phe Arg Cys Asn Leu Asp Glu Lys Leu Ile Arg Glu Thr
3S 40 45
GCC GAT GCA ATG GTA TCA AAG GGG CTT GCT GCA CTG GGA TAT AAG TAC 287
Ala Asp Ala Met Val Ser Lys Gly Leu Ala Ala Leu Gly Tyr Lys Tyr
ATC AAT CTT GAT GAC TGT TGG GCA GAA CTT AAC AGA GAT TCA CAG GGG 335
Ile Asn Leu Asp Asp Cys Trp Ala Glu Leu Asn Arg Asp Ser Gln Gly
AAT TTG GTT CCC AAA GGT TCA ACA TTC CCA TCA GGG ATC AAA GCC TTA 383
Asn Leu Val Pro Lys Gly Ser Thr Phe Pro Ser Gly Ile Lys Ala Leu
GCA GAT TAT GTT CAC AGC AAA GGC CTA AAG CTT GGA ATT TAC TCT GAT 431
Ala Asp Tyr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ser Asp
100 105 110
GCT GGA ACT CAG ACA TGT AGT AAA ACT ATG CCA GGT TCA TTA GGA CAC 479
Ala Gly Thr Gln Thr Cys Ser Lys Thr Met Pro Gly Ser Leu Gly His
115 120 125
GAA GAA CAA GAT GCC AAA ACC TTT GCT TCA TGG GGG GTA GAT TAC TTA 527
Glu Glu Gln Asp Ala Lys Thr Phe Ala Ser Trp Gly Val Asp Tyr Leu
130 135 140
AAG TAT GAC AAC TGT AAC AAC AAC AAC ATA AGC CCC AAG GAA AGG TAT 575
Lys Tyr Asp Asn Cys Asn Asn Asn Asn Ile Ser Pro Lys Glu Arg Tyr
145 150 155
CCA ATC ATG AGT AAA GCA TTG TTG AAC TCT GGA AGG TCC ATA TTT TTC 623
Pro I le Met Ser Lys Ala Leu Leu Asn Ser Gly Arg Ser I le Phe Phe
160 165 170
-
CA 022ll4l7 l997-07-2~
W 096/23869 PCTrUS96/01212
-28-
TCT CTA TGT GAA TGG GGA GAG GAA GAT CCA GCA ACA TGG GCA AAA GAA 671
Ser Leu Cys Glu Trp Gly Glu Glu Asp Pro Ala Thr Trp Ala Lys Glu
175 180 18S 190
GTT GGA AAC AGT TGG AGA ACC ACT GGA GAT ATA GAT GAC AGT TGG AGT 719
Val Gly Asn Ser Trp Arg Thr Thr Gly Asp Ile Asp Asp Ser Trp Ser
195 200 205
AGC ATG ACT TCT CGG GCA GAT ATG AAC GAC AAA TGG GCA TCT TAT GCT 767
Ser Met Thr Ser Arg Ala Asp Met Asn Asp Lys Trp Ala Ser Tyr Ala
210 215 220
GGT CCC GGT GGA TGG AAT GAT CCA GAC ATG TTG GAG GTG GGA AAT GGA 815
Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Glu Val Gly Asn Gly
215 220 225
GGC ATG ACT ACA ACG GAA TAT CGA TCC CAT TTG AGC ATT TGG GCA TTA 863
Gly Met Thr Thr Thr Glu Tyr Arg Ser His Phe Ser Ile Trp Ala Leu
230 235 240
GCA AAA GCA CCT CTA CTG ATT GGC TGT GAC ATT CGA TCC ATG GAC GGT 911
Ala Lys Ala Pro Leu Leu Ile Gly Cys Asp Ile Arg Ser Met Asp Gly
245 250 255 260
GCG ACT TTC CAA CTG CTA AGC AAT GCG GAA GTT ATT GCG GTT AAC CAA 9 59
Ala Thr Phe Gln Leu Leu Ser Asn Ala Glu Val Ile Ala Val Asn Gln
265 270 275
GAT AAA CTT GGC GTT CAA GGG AAC AAG GTT AAG ACT TAC GGA GAT TTG 1007
Asp Lys Leu Gly Val Gln Gly Asn Lys Val Lys Thr Tyr Gly Asp Leu
280 285 290
GAG GTT TGG GCT GGA CCT CTT AGT GGA AAG AGA GTA GCT GTC GCT TTG 1055
Glu Val Trp Ala Gly Pro Leu Ser Gly Lys Arg Val Ala Val Ala Leu
295 300 305
TGG AAT AGA GGA TCT TCC ACG GCT ACT ATT ACC GCG TAT TGG TCC GAC 1103
Trp Asn Arg Gly Ser Ser Thr Ala Thr Ile Thr Ala Tyr Trp Ser Asp
310 315 320
GTA GGC CTC CCG TCC ACG GCA GTG GTT AAT GCA CGA GAC TTA TGG GCG 1151
Val Gly Leu Pro Ser Thr Ala Val Val Asn Ala Arg Asp Leu Trp Ala
325 330 335 340
CAT TCA ACC GAA AAA TCA GTC AAA GGA CAA ATC TCA GCT GCA GTA GAT 1199
His Ser Thr Glu Lys Ser Val Lys Gly Gln Ile Ser Ala Ala Val Asp
345 350 355
GCC CAC GAT TCG AAA ATG TAT GTC CTA ACC CCA CAG TGA TTA ACA GGA 1247
Ala His Asp Ser Lys Met Tyr Val Leu Thr Pro Gln ***
370 375
GAA TGC AGA AGA CAA GTG ATG GTT GGC TCT TTC AAG GAT TTG ATT ACC 1295
TTA AAG AAT TTT TCA CAT GTT ATG AAT CAA TTC CAA GCA ATT ATG TGT 1343
CA 02211417 1997-07-2~
W 096/23869 PCTrUS96/01212 -29-
TTT GAA GAG ATT AAG TCA ATA AAT AGA AAA GTT ATT ATT GGA AAA AAA 1391
AAA AAA AAA AAA AAA AAA 1409
(3) INFORMATION FOR SEQ ID NO: 2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 378
(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: GREEN COFFEE BEAN
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2
Met Val Lys Ser Pro Gly Thr Glu Asp Tyr Thr Arg Arg Ser Leu Leu
1 5 10 15
~la Asn Gly Leu Gly Leu Thr Pro Pro Met Gly Trp Asn Ser Trp Asn
His Phe Arg Cys Asn Leu Asp Glu Lys Leu Ile Arg Glu Thr Ala Asp
Ala Met Val Ser Lys Gly Leu Ala Ala Leu Gly Tyr Lys Tyr Ile ~sn
Leu Asp Asp Cys Trp Ala Glu Leu Asn Arg Asp Ser Gln Gly Asn Leu
~al Pro Lys Gly Ser Thr Phe Pro Ser Gly Ile Lys Ala Leu Ala Asp
~yr Val His Ser Lys Gly Leu Lys Leu Gly Ile Tyr Ser Asp Ala Gly
100 105 110
Thr Gln Thr Cys Ser Lys Thr Met Pro Gly Ser Leu Gly His Glu Glu
115 120 125
Gln Asp Ala Lys Thr Phe Ala Ser Trp Gly Val Asp Tyr Leu Lys Tyr
130 135 140
Asp Asn Cys Asn Asn Asn Asn Ile Ser Pro Lys Glu Arg Tyr Pro Ile
145 150 155 160
Met Ser Lys Ala Leu Leu Asn Ser Gly Arg Ser Ile Phe Phe Ser Leu
165 170 175
CA 02211417 1997-07-2~
W 096/23869 PCTrUS96/01212
-30-
Cys Glu Trp Gly Glu Glu Asp Pro Ala Thr Trp Ala Lys Glu Val Gly
180 185 190
Asn Ser Trp Arg Thr Thr Gly Asp Ile Asp Asp Ser Trp Ser Ser Met
195 200 205
Thr Ser Arg Ala Asp Met Asn Asp Lys Trp Ala Ser Tyr Ala Gly Pro
210 215 220
Gly Gly Trp Asn Asp Pro Asp Met Leu Glu Val Gly Asn Gly Gly Met
225 230 235 240
~hr Thr Thr Glu Tyr Arg Ser His Phe Ser Ile Trp Ala Leu Ala Lys
245 250 255
~la Pro Leu Leu Ile Gly Cys Asp Ile Arg Ser Met Asp Gly Ala Thr
260 265 270
Phe Gln Leu Leu Ser Asn Ala Glu Val Ile Ala Val Asn Gln Asp Lys
275 280 285
Leu Gly Val Gln Gly Asn Lys Val Lys Thr Tyr Gly Asp Leu Glu Val
290 295 300
Trp Ala Gly Pro Leu Ser Gly Lys Arg Val Ala Val Ala Leu Trp Asn
305 310 315 320
~rg Gly Ser Ser Thr Ala Thr Ile Thr Ala Tyr Trp Ser Asp Val Gly
325 330 335
~eu Pro Ser Thr Ala Val Val Asn Ala Arg Asp Leu Trp Ala His Ser
340 345 350
~hr Glu Lys Ser Val Lys Gly Gln Ile Ser Ala Ala Val Asp Ala His
355 360 365
Asp Ser Lys Met Tyr Val Leu Thr Pro Gln
370 375
(4) INFORMATION FOR SEQ ID NO: 3
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 411
(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: CYAMOPSIS TETRAGONOLOBA
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
CA 02211417 1997-07-2~
W096/23869 PCT~S96101212
-31-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3
Met Ala Thr His Tyr Ser Ile Ile Gly Gly Met Ile Ile Val Val Leu
1 5 10 15
~eu Met Ile Ile Gly Ser Glu Gly Gly Arg Leu Leu Glu Lys Lys Asn
~rg Thr Ser Ala Glu Ala Glu His Tyr Asn Val Arg Arg Tyr Leu Ala
Glu Asn Gly Leu Gly Gln Thr Pro Pro Met Gly Trp Asn Ser Trp Asn
His Phe Gly Cys Asp Ile Asn Glu Asn Val Val Arg Glu Thr Ala Asp
~la Met Val Ser Thr Gly Leu Ala Ala Leu Gly Tyr Gln Tyr Ile Asn
~eu Asp Asp Cys Trp Ala Glu Leu Asn Arg Asp Ser Glu Gly Asn Met
100 105 110
Val Pro Asn Ala Ala Ala Phe Pro Ser Gly Ile Lys Ala Leu Ala Asp
115 120 125
Tyr Val His Ser Lys Gly Leu Lys Leu Gly Val Tyr Ser Asp Ala Gly
130 135 140
Asn Gln Thr Cys Ser Lys Arg Met Pro Gly Ser Leu Gly His Glu Glu
145 150 155 160
~ln Asp Ala Lys Thr Phe Ala Ser Trp Gly Val Asp Tyr Leu Lys Tyr
165 170 175
~sp Asn Cys Glu Asn Leu Gly Ile Ser Val Lys Glu Arg Tyr Pro Pro
180 185 190
Met Gly Lys Ala Leu Leu Ser Ser Gly Arg Pro Ile Phe Phe Ser Met
195 200 205
Cys Glu Trp Gly Trp Glu Asp Pro Gln Ile Trp Ala Lys Ser Ile Gly
210 215 220
Asn Ser Trp Arg Thr Thr Gly Asp Ile Glu Asp Asn Trp Asn Ser Met
225 230 235 240
~hr Ser Ile Ala Asp Ser Asn Asp Lys Trp Ala Ser Tyr Ala Gly Pro
245 250 255
~ly Gly Trp Asn Asp Pro Asp Met Leu Glu Val Gly Asn Gly Gly Met
260 265 270
~hr Thr Glu Glu Tyr Arg Ser His Phe Ser Ile Trp Ala Leu Ala Lys
275 280 285
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Ala Pro Leu Leu Val Gly Cys Asp Ile Arg Ala Met Asp Asp Thr Thr
290 295 300
His Glu Leu Ile Ser Asn Ala Glu Val Ile Ala Val Asn Gln Asp Lys
305 310 315 320
~eu Gly Val Gln Gly Lys Lys Val Lys Ser Thr Asn Asp Leu Glu Val
325 330 335
~rp Ala Gly Pro Leu Ser Asp Asn Lys Val Ala Val Ile Leu Trp Asn
340 345 350
Arg Ser Ser Ser Arg Ala Thr Val Thr Ala Ser Trp Ser Asp Ile Gly
355 360 365
Leu Gln Gln Gly Thr Thr Val Asp Ala Arg Asp Leu Trp Glu His Ser
370 375 380
Thr Gln Ser Leu Val Ser Gly Glu Ile Ser Ala Glu Ile Asp Ser His
385 390 395 400
Ala Cys Lys Met Tyr Val Leu Thr Pro Arg Ser
405 410
(5) INFORMATION FOR SEQ ID NO: 4
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 429
(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HUMAN PLACENTA
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4
Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu
1 5 10 15
~rg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu
Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu
Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile
Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly
CA 02211417 1997-07-2~
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~rp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys I le Asp Asp Cys Trp Met
~la Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg
100 105 110
Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly
115 120 125
Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly
130 135 140
Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala
145 150 155 160
~sp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser
165 170 175
~eu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn
180 185 190
Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met
195 200 205
Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn
210 215 220
His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys
225 230 235 240
~er Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val
245 250 255
~la Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn
260 265 270
Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala
275 280 285
Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser
290 295 300
Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn
305 310 315 320
~ln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn
325 330 335
~he Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala
340 345 350
Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala
355 360 365
Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile
370 375 380
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Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr
385 390 395 400
Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln
405 410 415
~eu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu
420 425
~6) INFORMATION ~OR SEQ ID NO: 5
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 432
(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: SACCHAROMYCES CEREVISIAE
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5
Met Phe Ala Phe Tyr Phe Leu Thr Ala Cys Ile Ser Leu Lys Gly Val
1 5 10 15
~he Gly Val Ser Pro Ser Tyr Asn Gly Leu Gly Leu Thr Pro Gln Met
~ly Trp Asp Asn Trp Asn Thr Phe Ala Cys Asp Val Ser Glu Gln Leu
Leu Leu Asp Thr Ala Asp Arg Ile Ser Asp Leu Gly Leu Lys Asp Met
Gly Tyr Lys Tyr Ile Ile Leu Asp Asp Cys Trp Ser Ser Gly Arg Asp
~er Asp Gly Phe Leu Val Ala Asp Glu Gln Lys Phe Pro Asn Gly Met
~ly His Val Ala Asp His Leu His Asn Asn Ser Phe Leu Phe Gly Met
100 105 110
~yr Ser Ser Ala Gly Glu Tyr Thr Cys Ala Gly Tyr Pro Gly Ser Leu
115 120 125
Gly Arg Glu Glu Glu Asp Ala Gln Phe Phe Ala Asn Asn Arg Val Asp
130 135 140
Tyr Leu Lys Tyr Asp Asn Cys Tyr Asn Lys Gly Gln Phe Gly Thr Pro
145 150 155 160
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~lu Ile Ser Tyr His Arg Tyr Lys Ala Met Ser Asp Ala Leu Asn Lys
165 17û 175
~hr Gly Arg Pro I le Phe Tyr Ser Leu Cys Asn Trp Gly Gln Asp Leu
180 185 190
Thr Phe Tyr Trp Gly Ser Gly Ile Ala Asn Ser Trp Arg Met Ser Gly
195 200 205
Asp Val Thr Ala Glu Phe Thr Arg Pro Asp Ser Arg Cys Pro Cys Asp
210 215 220
Gly Asp Glu Tyr Asp Cys Lys Tyr Ala Gly Phe His Cys Ser Ile Met
225 230 235 240
~sn Ile Leu Asn Lys Ala Ala Pro Net Gly Gln Asn Ala Gly Val Gly
245 250 255
~ly Trp Asn Asp Leu Asp Asn Leu Glu Val Gly Val Gly Asn Leu Thr
260 265 270
Asp Asp Glu Glu Lys Ala His Phe Ser Met Trp Ala Met Val Lys Ser
275 280 285
Pro Leu Ile Ile Gly Ala Asn Val Asn Asn Leu Lys Ala Ser Ser Tyr
290 295 300
Ser Ile Tyr Ser Gln Ala Ser Val Ile Ala Ile Asn Gln Asp Ser Asn
305 310 315 320
~ly Ile Pro Ala Thr Arg Val Trp Arg Tyr Tyr Val Ser Asp Thr Asp
325 330 335
~lu Tyr Gly Gln Gly Glu Ile Gln Met Trp Ser Gly Pro Leu Asp Asn
340 345 350
Gly Asp Gln Val Val Ala Leu Leu Asn Gly Gly Ser Val Ser Arg Pro
355 360 365
Met Asn Thr Thr Leu Glu Glu I le Phe Phe Asp Ser Asn Leu Gly Ser
370 375 380
Lys Lys Leu Thr Ser Thr Trp Asp Ile Tyr Asp Leu Trp Ala Asn Arg
385 390 395 400
~al Asp Asn Ser Thr Ala Ser Ala Ile Leu Gly Arg Asn Lys Thr Ala
405 410 415
~hr Gly Ile Leu Tyr Asn Ala Thr Glu Gln Ser Tyr Lys Asp Gly Leu
420 425 430
(7) INFORMATION FOR SEQ ID NO: 6
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441
CA 022ll4l7 l997-07-2~
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(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PROTEIN
(iii) HYPO~ CAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ASPERGILLUS NIGER
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(xi) SE~UENCE DESCRIPTION: SEQ ID NO: 6
Met Ile Gln Gly Leu Glu Ser Ile Met Asn Gln Gly Thr Lys Arg Ile
1 5 10 15~eu Leu Ala Ala Thr Leu Ala Ala Thr Pro Trp Gln Val Tyr Gly Ser
~le Glu Gln Pro Ser Leu Leu Pro Thr Pro Pro Met Gly Phe Asn Asn
Trp Ala Arg Phe Met Cys Asp Leu Asn Glu Thr Leu Phe Thr Glu Thr
Ala Asp Thr Met Ala Ala Asn Gly Leu Arg Asp Ala Gly Tyr Asn Arg
~le Asn Leu Asp Asp Cys Trp Met Ala Tyr Gln Arg Ser Asp Asn Gly
~er Leu Gln Trp Asn Thr Thr Lys Phe Pro His Gly Leu Pro Trp Leu
100 105 110
~la Lys Tyr Val Lys Ala Lys Gly Phe His Phe Gly Ile Tyr Glu Asp
115 120 125
Ser Gly Asn Met Thr Cys Gly Gly Tyr Pro Gly Ser Tyr Asn His Glu
130 135 140
Glu Gln Asp Ala Asn Thr Phe Ala Ser Trp Gly Ile Asp Tyr Leu Lys
145 150 155 160
~eu Asp Gly Cys Asn Val Tyr Ala Thr Gln Gly Arg Thr Leu Glu Glu
165 170 175
~lu Tyr Lys Gln Arg Tyr Gly His Trp His Gln Val Leu Ser Lys Met
180 185 190
~ln His Pro Leu Ile Phe Ser Glu Ser Ala Pro Ala Tyr Phe Ala Gly
195 200 205
~hr Asp Asn Asn Thr Asp Trp Tyr Thr Val Met Asp Trp Val Pro Ile
210 215 220
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Tyr Gly Glu Leu Ala Arg His Ser Thr Asp Ile Leu Val Tyr Ser Gly
225 230 235 240
Ala Gly Ser Ala Trp Asp Ser Ile Met Asn Asn Tyr Asn Tyr Asn Thr
245 250 255
~eu Leu Ala Arg Tyr Gln Arg Pro Gly Tyr Phe Asn Asp Pro Asp Phe
260 265 270
~eu Ile Pro Asp His Pro Gly Leu Thr Ala Asp Glu Lys Arg Ser His
275 280 285
Phe Ala Leu Trp Ala Ser Phe Ser Ala Pro Leu Ile Ile Ser Ala Tyr
290 295 300
Ile Pro Ala Leu Ser Lys Asp Glu Ile Ala Phe Leu Thr Asn Glu Ala
305 310 315 320
~eu Ile Ala Val Asn Gln Asp Pro Leu Ala Gln Gln Ala Thr Leu Ala
325 330 335
~er Arg Asp Asp Thr Leu Asp Ile Leu Thr Arg Ser Leu Ala Asn Gly
340 345 350
~sp Arg Leu Leu Thr Val Leu Asn Lys Gly Asn Thr Thr Val Thr Arg
355 360 365
Asp Ile Pro Val Gln Trp Leu Gly Leu Thr Glu Thr Asp Cys Thr Tyr
370 375 380
Thr Ala Glu Asp Leu Trp Asp Gly Lys Thr Gln Lys Ile Ser Asp His
385 390 395 400
~le Lys Ile Glu Leu Ala Ser His Ala Thr Ala Val Phe Arg Leu Ser
405 410 415
~eu Pro Gln Gly Cys Ser Ser Val Val Pro Thr Gly Leu Val Phe Asn
420 425 430
~hr Ala Ser Gly Asn Cys Leu Thr Ala
435 440
~8) INFORMATION FOR SEQ ID NO: 7
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: UNKNOWN
(ii) MOLECULE TYPE:
(A) DESCRIPTION: OLIGONUCLEOTIDE
(iii) HYPOTHETICAL: NO
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(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: GREEN COFFEE BEAN
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7
ACACCWCCWA TGGMTGGA 18
(9) INFORMATION FOR SEQ ID NO: 8
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: UNKNOWN
(ii) MOLECULE TYPE:
(A) DESCRIPTION: OLIGONUCLEOTIDE
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: GREEN COFFEE BEAN
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8
TGWGGDGTNA GVACRTACAT 20
(10) INFORMATION FOR SEQ ID NO: 9
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: UNKNOWN
(ii) MOLECULE TYPE:
(A) DESCRIPTION: OLIGONUCLEOTIDE
(iii~ HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: GREEN COFFEE BEAN
(B) INDIVIDUAL ISOLATE: ALPHA-GALACTOSIDASE
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9
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GACTCGAGTC GACATCGA 18
(11) INFORMATION FOR SEQ ID NO: 10
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66
(B) TYPB: NUCLEIC ACID
(C) STRANDEDNESS: DOUBLE
(D) TOPOLOGY: UNKNOWN
(ii) MOLECULE TYPE:
(A) DESCRIPTION: OLIGONUCLEOTIDE
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(B) INDIVIDUAL ISOLATE: SIGNAL SEQUENCE OF ALPHA
MATING FACTOR AND N-TERMINAL OF
ALPHA-GALACTOSIDASE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10
GGGGTATCTC TCGAGAAAAG AGAGGCTGAA GCTTACGTAG AATCCTTAGC 50
AAATGGGCTT GGTCTA 66
(12) INFORMATION FOR SEQ ID NO: 11
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: AMINO ACID
(ii) MOLECULE TYPE:
(A) DESCRIPTION: PEPTIDE
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(B) INDIVIDUAL ISOLATE: SIGNAL SEQUENCE OF ALPHA
MATING FACTOR AND N-TERMINAL OF
ALPHA-GALACTOSIDASE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11
Val Set Leu Glu Lys Lys Arg Glu Ala Glu Ala Tyr Val Glu Phe
1 5 10 15
Leu Ala Asn Gly Leu Gly Leu
