Gene encoding endoglycoceramidase activator

An isolated DNA having a sequence encoding a polypeptide possessing endoglycoceramidase activator activity or functionally equivalent variants thereof; and a method for producing a polypeptide possessing endoglycoceramidase activator activity or functionally equivalent variants thereof by gene recombinant technology.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a DNA encoding a polypeptide which 
activates endoglycoceramidase, an enzyme useful for structural, functional 
and other analyses of glycolipids in sugar chain engineering, or which 
possesses endoglycoceramidase activator activity. The present invention 
also relates to an industrial production method for a polypeptide 
possessing endoglycoceramidase activator activity using a recombinant 
incorporating a recombinant plasmid having the DNA inserted therein. 
2. Discussion of the Related Art 
Endoglycoceramidase (EC3.2.1.123), an enzyme first isolated from the 
Actinomycetes of Rhodococcus strain The Journal of Biological Chemistry 
261, 14278-14282 (1986)! hydrolyzes the glycoside linkage between the 
sugar chain and ceramide in glycosphingolipid to liberate the sugar chain 
and ceramide in their complete form. Rhodococcus strains are also known to 
produce three different types of endoglycoceramidase (I, II, III) of 
different substrate specificities The Journal of Biological Chemistry 264 
(16) 9510-9519 (1989)!. Other known types of endoglycoceramidase include 
leech-derived endoglycoceramidase (ceramide-glycanase)Biochemical and 
Biophysical Research Communications 141, 346-352 (1986)!. Although these 
enzymes are capable of efficiently decomposing glycolipids in the presence 
of various surfactants, glycolipid decomposition by them in the absence of 
surfactants is very slow. 
On the other hand, the endoglycoceramidase-producing Rhodococcus strain 
produce two proteinic activators (activator I, activator II) which 
activate endoglycoceramidase with molecular specificity to allow 
endoglycoceramidase to hydrolyze glycolipids even in the absence of 
surfactants, at the same time with the endoglycoceramidase production. 
Activator I activates endoglycoceramidase I, while activator II activates 
endoglycoceramidase II. It is a well-known fact, however, that activator 
II activates endoglycoceramidase II more potently than endoglycoceramidase 
I, although it activates endoglycoceramidase I as well, and that activator 
II is also capable of activating leech-derived endoglycoceramidase 
(ceramide-glycanase) The Journal of Biological Chemistry 266 (12) 
7919-7926 (1991)!. 
Also, activator II, which has a molecular weight of 69.2 kDa, has been 
shown to be digested to 27.9 kDa by complete digestion with trypsin, while 
retaining its activity in an intact state The Journal of Biochemistry 
110, 328-332 (1991)!. In the presence of activator II, the optimum pH for 
endoglycoceramidase II shifts toward the neutral side, making it possible 
for endoglycoceramidase II to hydrolyze glycolipids even at pH 7.5. These 
facts suggest that the use of activator II may permit endoglycoceramidase 
II to exert its action on viable cells under physiological conditions 
(nearly neutral pH, in the absence of surfactants) under which the action 
of the enzyme is otherwise hampered. In other words, the use of activator 
II has been proven to enable the analysis of intracellular function of 
glycolipids by use of endoglycoceramidase II. 
It should be noted, however, that the action of endoglycoceramidase II on 
viable cells requires large amounts of high-purity endoglycoceramidase II 
and endoglycoceramidase activator II. Production of endoglycoceramidase 
activator II using the producer Rhodococcus requires long cultivation time 
and many steps of purification; it is very difficult to obtain large 
amounts of high-purity activator II. There is therefore need for a method 
of producing the enzyme at lower cost and higher purity. 
Also, because the amino acid sequence and gene structure of 
endoglycoceramidase activator II remain unknown, it is difficult to 
produce endoglycoceramidase activator II by gene engineering technology. 
Accordingly, an objective of the present invention is to provide a DNA 
encoding a polypeptide possessing endoglycoceramidase activator activity. 
Another objective of the present invention is to provide a method of 
producing an endoglycoceramidase activator by gene engineering technology. 
SUMMARY OF THE INVENTION 
In order to achieve the above object, the present inventors conducted 
intensive studies in an effort to isolate the DNA encoding a polypeptide 
possessing endoglycoceramidase activator activity for elucidation of its 
nucleotide sequence. As a result, the present inventors at last succeeded 
in isolating a DNA encoding a polypeptide possessing endoglycoceramidase 
activator activity, and in elucidating the nucleotide sequence of the gene 
structure involved in the activity. Also, they succeeded in expressing the 
DNA in an organism to which a plasmid vector carrying the DNA is 
introduced. Based upon these facts, the present invention has been 
completed. 
In one embodiment, the present invention relates to an isolated DNA having 
a sequence encoding a polypeptide possessing endoglycoceramidase activator 
activity or functionally equivalent variants thereof. Specifically, the 
isolated DNA comprises a DNA sequence selected from the group consisting 
of (a) to (d): 
(a) a DNA sequence of SEQ ID NO:2 or SEQ ID NO:4, or a fragment thereof; 
(b) a DNA sequence encoding an amino acid sequence of SEQ ID NO:1 or SEQ ID 
NO:3, or a fragment thereof; 
(c) a DNA sequence encoding an amino acid sequence resulting from deletion, 
addition, insertion or substitution of one or more amino acids in the 
amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, or a fragment thereof; 
and 
(d) a DNA sequence capable of hybridizing to any one of (a) to (c) above. 
In another embodiment, the present invention relates to a recombinant DNA 
which comprises the isolated DNA of the present invention, to a vector 
comprising the recombinant DNA, and to a cell of a procaryote or eucaryote 
transformed with the vector. 
In another embodiment, the present invention relates to a method for 
producing a polypeptide possessing endoglycoceramidase activator activity 
or functionally equivalent variants thereof, comprising the steps of: 
(a) culturing the cell of the present invention; and 
(b) recovering the polypeptide possessing endoglycoceramidase activator 
activity or functionally equivalent variants thereof from the culture 
obtained in Step (a). 
In another embodiment, the present invention relates to a polypeptide 
possessing endoglycoceramidase activator activity or functionally 
equivalent variants thereof produced by the present method or encoded by 
the isolated DNA of the present invention. 
In another embodiment, the present invention relates to a synthetic 
oligonucleotide probe or primer which specifically hybridizes with the 
isolated DNA of the present invention. 
In another embodiment, the present invention relates to an antibody or 
fragment thereof which specifically binds the polypeptide of the present 
invention. 
The amino acid sequence involved in the activity of endoglycoceramidase 
activator and its nucleotide sequence have first been established by the 
present invention, thereby providing the gene of endoglycoceramidase 
activator and an industrially advantageous method for producing a 
polypeptide possessing endoglycoceramidase activator activity by gene 
engineering techniques. 
By use of the DNA sequence encoding endoglycoceramidase activator of the 
present invention, it becomes possible to probe DNA which has a different 
sequence from the present invention but possibly encodes a polypeptide 
possessing a functionally equivalent activity to the present invention. 
Also, the amino acid sequence corresponding to the DNA sequence encoding 
endoglycoceramidase activator of the present invention is useful for the 
preparation of the antibody to the endoglycoceramidase activator of the 
present invention.

DETAILED DESCRIPTION OF THE INVENTION 
Endoglycoceramidase activator as mentioned herein refers to a protein which 
activates endoglycoceramidase in the absence of surfactant. 
Endoglycoceramidase activator activity as mentioned herein can be 
determined as follows: 
The activity can be determined by the method described in The Journal of 
Biological Chemistry 266 (12) 7919-7926 (1991), using an extract from 
recombinant Escherichia coli cells. Specifically, 40 nmol of asialo-GM1, 5 
.mu.g of bovine serum albumin, 0.3 milliunits of purified 
endoglycoceramidase and an appropriate amount of a crude extract 
(endoglycoceramidase activator protein) are mixed in 40 .mu.l of a 20 mmol 
sodium acetate buffer (pH 5.5), previously isotonized with 0.85% NaCl , to 
yield a standard test mixture. Enzyme activity is then determined by the 
method described in The Journal of Biological Chemistry 264, 9510-9519 
(1989). After incubation at 37.degree. C. for 60 minutes, the reaction is 
terminated by the addition of 250 .mu.l of a carbonate-cyanide solution 
(pH 11); reducing activity is assessed by the method of Park and Johnson 
The Journal of Biological Chemistry 181, 149-151 (1949)!. For control 
experiment, the substrate and crude extract (endoglycoceramidase activator 
protein), or the substrate and endoglycoceramidase, are separately 
incubated. The sum of the both control values is subtracted from the above 
actual sample value. One unit of endoglycoceramidase activator protein is 
defined as the amount of endoglycoceramidase activator protein which 
increases the hydrolysis of asialo-GM1 by 1 .mu.mol per minute under the 
above-described standard experimental conditions. 
The term "a polypeptide possessing endoglycoceramidase activator activity" 
as used in the present specification includes not only those having an 
amino acid sequence of native endoglycoceramidase activator but also its 
variants due to modification of amino acid sequence by, for example, 
deletion, substitution, insertion, or addition of amino acid(s), as long 
as they are capable of activating endoglycoceramidase. "Native 
endoglycoceramidase activator" used herein includes, but is not limited 
to, those produced by Rhodococcus strains. Also included are those derived 
from other microorganisms, such as other Actinomycetes, bacteria, yeasts, 
fungi, Ascomycetes, and Basidiomycetes, and those derived from plants, 
animals, insects, and other living things. 
The term "functionally equivalent variant" as used herein is defined as 
follows: 
A naturally-occurring protein can undergo amino acid deletion, insertion, 
addition, substitution and other variations in its amino acid sequence due 
to modifications, etc. of the protein itself in vivo or during 
purification, as well as those due to polymorphism and variation of the 
gene encoding it. It is a well-known fact that there are some such 
polypeptides which are substantially equivalent to variation-free proteins 
in terms of physiological and biological activity. A polypeptide which is 
structurally different from the corresponding protein but has no 
significant functional difference from the protein is referred to as a 
functionally equivalent variant. 
The same applies to polypeptides prepared by artificially introducing such 
variations into the amino acid sequence of a protein. Although more 
diverse variants can be thus obtained, the resulting variants are 
construed as functionally equivalent variants, as long as their 
physiological activity is substantially equivalent to that of the original 
variation-free protein. 
For example, the methionine residue at the N-terminus of a protein 
expressed in Escherichia coli is reportedly often removed by the action of 
methionine aminopeptidase, but some such expressed proteins have the 
methionine residue and others do not. However, the presence or absence of 
the methionine residue does not affect protein activity in most cases. It 
is also known that a polypeptide resulting from replacement of a 
particular cysteine residue with serine in the amino acid sequence of 
human interleukin 2 (IL-2) retains IL-2 activity Science, 224, 1431 
(1984)!. 
In addition, in producing a protein by gene engineering, the desired 
protein is often expressed as a fused protein. For example, the N-terminal 
peptide chain derived from another protein is added to the N-terminus of 
the desired protein to enhance the expression of the desired protein, or 
purification of the desired protein is facilitated by adding an 
appropriate peptide chain to the N- or C-terminus of the desired protein, 
expressing the protein, and using a carrier showing affinity for the 
peptide chain added. 
Also, with regards to the codon (triplet base combination) determining a 
particular amino acid on the gene, 1 to 6 kinds are known to exist for 
each amino acid. Therefore, there can be a large number of genes encoding 
an amino acid sequence, though depending on the amino acid sequence. In 
nature, the gene is not stable, commonly undergoing nucleic acid 
variation. A variation on the gene may not affect the amino acid sequence 
to be encoded (silent variation); in this case, it can be said that a 
different gene encoding the same amino acid sequence has been produced. 
The possibility is therefore unnegligible that even when a gene encoding a 
particular amino acid sequence is isolated, a variety of genes encoding 
the same amino acid sequence are produced with generation passage of the 
organism containing it. 
Moreover, it is not difficult to artificially produce a variety of genes 
encoding the same amino acid sequence by means of various gene engineering 
techniques. 
For example, when a codon used in the natural gene encoding the desired 
protein is low in availability in the host used to produce the protein by 
gene engineering, the amount of protein expressed is sometimes 
insufficient. In this case, expression of the desired protein is enhanced 
by artificially converting the codon into another one of high availability 
in the host without changing the amino acid sequence encoded. It is of 
course possible to artificially produce a variety of genes encoding a 
particular amino acid sequence. Such artificially produced different 
polynucleotides are therefore included in the scope of the present 
invention, as long as an amino acid sequence disclosed herein is encoded. 
Additionally, a polypeptide resulting from at least one change, such as 
deletion, addition, insertion or substitution, of one or more amino acid 
residues in the amino acid sequence of the desired protein commonly 
possesses an activity functionally equivalent to that of the desired 
protein; genes encoding such polypeptides are also included in the scope 
of the present invention, whether isolated from natural sources or 
produced artificially. 
In general, functionally equivalent variants are often homologous to each 
other in terms of genes encoding them. Nucleic acid molecules capable of 
hybridizing to a gene of the present invention, and encoding a polypeptide 
possessing endoglycoceramidase activator activity, are therefore also 
included in the scope of the present invention. 
The present invention is hereinafter described in detail as to isolation 
and sequencing of the DNA of the present invention with reference to 
endoglycoceramidase activator II. 
First, information regarding a partial amino acid sequence of purified 
endoglycoceramidase activator II is obtained. Specifically, 
endoglycoceramidase activator II as purified by the method described in 
The Journal of Biochemistry 110, 328-332 (1991), for instance, is directly 
subjected to the Edman degradation method The Journal of Biological 
Chemistry 256, 7990-7997 (1981)! for amino acid sequencing by a 
conventional method. Alternatively, said activator may be partially 
hydrolyzed by the action of a protein hydrolase of high specificity, the 
resulting peptide fragment may be separated, purified and subjected to 
amino acid sequencing to determine its internal partial amino acid 
sequence. 
On the basis of the thus-obtained partial amino acid sequence information, 
the endoglycoceramidase activator II gene is cloned. For this purpose, a 
commonly used PCR or hybridization method can be used. 
Next, on the basis of the partial amino acid sequence information, 
synthetic oligonucleotides are designed for use as Southern hybridization 
probes. Separately, the genomic DNA of Rhodococcus sp. M-777 is completely 
digested with the appropriate restriction enzymes including MluI, SalI, 
PstI, and BamHI and subjected to agarose gel electrophoresis for 
separation Molecular Cloning, A Laboratory Manual, 2nd ed., T. Maniatis 
et al., Chapter 6, 3-20, Cold Spring Harbor Laboratory Press, (1989)!, and 
the separated fragments are blotted onto a nylon membrane by a 
conventional method Molecular Cloning, A Laboratory Manual, 2nd ed., T. 
Maniatis et al., Chapter 9, 34, Cold Spring Harbor Laboratory Press, 
(1989)!. 
Hybridization can be conducted under commonly used conditions. For example, 
the nylon membrane is blocked at 65.degree. C. in a prehybridization 
solution containing 6.times.SSC (1 .times.SSC is prepared by dissolving 
8.77 g NaCl and 4.41 g sodium citrate in 1 L of water), 0.5% SDS, 5 
.times. Denhardt's solution and 100 .mu.g/ml salmon sperm DNA, and each 
.sup.32 P-labeled synthetic oligonucleotide was added, followed by 
overnight incubation at 65.degree. C. After the nylon membrane is washed 
in 2 .times. SSC containing 0.1% SDS at 55.degree. C. for 30 minutes, an 
autoradiogram is taken to detect a DNA fragment that hybridizes to the 
synthetic oligonucleotide probe. After the DNA fragment corresponding to 
the band detected is extracted and purified from the gel, the DNA fragment 
is inserted into a plasmid vector by a commonly used method. Useful 
plasmid vectors include, but are not limited to, commercially available 
pUC18, pUC19, pUC119 and pTV118N (all are products of Takara Shuzo). 
Then, thus-obtained recombinant plasmid is introduced into a host to 
transform the host. Usable host cells include procaryotic cells of 
bacteria (e.g., Escherichia coli) and Actinomyces, and eucaryotic cells of 
yeast, fungi, animals, plants, etc. 
When the host is Escherichia coli, it may be of a wild strain or a variant 
strain, as long as it is capable of being transformed and expressing a 
gene. This plasmid introduction can be achieved by a commonly used method, 
such as the method described in Molecular Cloning, A Laboratory Manual 
2nd. ed., T. Maniatis et al., Chapter 1, 74-84, Cold Spring Harbor 
Laboratory Press (1989). 
Next, a transformant harboring the desired DNA fragment is selected. For 
this purpose, the characteristics of the plasmid vector are utilized. In 
the case of pUC19, for instance, colonies having a foreign gene introduced 
thereto are selected by selecting ampicillin-resistant colonies on an 
ampicillin-containing plate, or selecting ampicillin-resistant white 
colonies on a plate containing ampicillin, 5-bromo-4-chloro-3-indolyl- 
.beta.-D-galactoside (X-Gal) and isopropyl-.beta.-D-thiogalactopyranoside 
(IPTG). 
Next, the colony having a vector containing the desired DNA fragment is 
then selected out of the above population. This selection is achieved by 
using colony hybridization Molecular Cloning, A Laboratory Manual, 2nd 
ed., T. Maniatis et al., Chapter 1, 90-104, Cold Spring Harbor Laboratory 
Press (1989)! or plaque hybridization Molecular Cloning, A Laboratory 
Manual, 2nd ed., T. Maniatis et al., Chapter 2, 108-117, Cold Spring 
Harbor Laboratory Press (1989)!, chosen appropriately according to vector 
types. PCR methods Molecular Cloning, A Laboratory Manual, 2nd ed., T. 
Maniatis et al., Chapter 14, 15-19, Cold Spring Harbor Laboratory Press 
(1989)! are also applicable. 
Once the vector containing the obtained DNA fragment is selected, the base 
sequence of the obtained DNA fragment inserted in this vector is 
determined by an ordinary method, such as the dideoxy chain terminator 
method Molecular Cloning, A Laboratory Manual, 2nd ed., T. Maniatis et 
al., Chapter 13, 3-10, Cold Spring Harbor Laboratory Press (1989)!. The 
thus-determined base sequence is compared with the N-terminal sequence, 
partial amino acid sequence, molecular weight, etc. of endoglycoceramidase 
activator II to know the gene structure and entire amino acid sequence of 
endoglycoceramidase activator II. When the obtained DNA fragment does not 
contain the full-length endoglycoceramidase activator II gene, the 
full-length endoglycoceramidase activator II gene can be obtained by 
digesting genomic DNA of Rhodococcus sp. M-777 with other restriction 
enzymes, obtaining the lacking portion from the digests by hybridization, 
etc. using a part of the DNA fragment obtained above as a probe, then 
joining the lacking portion. 
The entire or part of the resulting endoglycoceramidase activator II gene 
as obtained above is inserted into an appropriate plasmid vector, which is 
then transformed into a host cell. The transformant thus obtained is 
cultured under commonly used conditions to produce a polypeptide 
possessing endoglycoceramidase activator II activity. 
For example, when Escherichia coli and pET23b produced by Novagen! are 
used as a host cell and plasmid vector, respectively, said transformant is 
cultured at 37.degree. C. overnight in an L medium (0.1% Trypton, 0.05% 
yeast extract, 0.1% NaCl, pH 7.2) containing 100 .mu.g/ml ampicillin; upon 
reach of an absorbance at 600 nm of about 0.5, IPTG is added, followed by 
further overnight shaking culture at 37.degree. C. After completion of the 
cultivation, cells are recovered, disrupted by ultrasonication etc. and 
centrifuged. The resulting supernatant is subjected to an ordinary protein 
purification process as described below to yield high-purity 
endoglycoceramidase activator II. There may be the case where the 
polypeptide expressed can be produced in the form of inclusion body. 
The expression of the gene product can be confirmed by, for example, 
determining endoglycoceramidase activator II activity. When the 
recombinant is Escherichia coli, for example, activity can be determined 
by the method described in the Journal of Biological Chemistry, 266 (12) 
7919-7929 (1991), using the extract of the recombinant Escherichia coli as 
an enzyme solution. Specifically, since endoglycoceramidase II requires a 
surfactant, such as Triton X-100, for expression of its activity, 
endoglycoceramidase activator II activity can be assessed by measuring the 
increment of endoglycoceramidase II activity in the absence of surfactants 
caused by the addition of a crude extract. 
Specifically, 40 nmol asialo-GM1 (produced by IATRON), 5 .mu.g of bovine 
serum albumin, 0.3 milliunits of purified endoglycoceramidase II and an 
appropriate amount of the crude extract (endoglycoceramidase activator II 
protein) are mixed in 40 .mu.l of a 20 mmol sodium acetate buffer (pH 
5.5), previously isotonized with 0.85% NaCl, to yield a standard test 
mixture. Enzyme activity is then determined by the method described in The 
Journal of Biological Chemistry 264, 9510-9519 (1989). After incubation at 
37.degree. C. for 60 minutes, the reaction is terminated by the addition 
of 250 .mu.l of a carbonate-cyanide solution (pH 11); reducing activity is 
assessed by the method of Park and Johnson The Journal of Biological 
Chemistry 181, 149-151 (1949)!. For control experiment, the substrate and 
crude extract (endoglycoceramidase activator II protein), or the substrate 
and endoglycoceramidase, are separately incubated. The sum of the both 
control values is subtracted from the above actual sample value. One unit 
of endoglycoceramidase activator II protein is defined as the amount of 
said protein which increases the hydrolysis of asialo-GM1 by 1 .mu.mol per 
minute under the above-described standard experimental conditions. 
Expression can also be confirmed immunologically using an antiserum 
obtained by immunizing a rat with purified endoglycoceramidase activator 
II. 
When the desired expression of endoglycoceramidase activator II is noted, 
optimum conditions for endoglycoceramidase activator II expression are 
selected. 
Endoglycoceramidase activator II can be purified from the transformant 
culture by an ordinary method. That is, the transformant cells are 
collected by centrifugation, disrupted by ultrasonication, or the like, 
and then subjected to centrifugation, etc. to yield a cell-free extract, 
which can be purified by common protein purification methods, such as 
salting-out and various chromatographies including ion exchange, gel 
filtration, hydrophobic interaction and affinity chromatographies. 
Depending on the host-vector system used, the expression product is 
extracellularly secreted by the transformant; in this case, the product 
can be purified from the culture supernatant in the same manner as that 
described above. When the host is Escherichia coli, the expression product 
is sometimes formed as an insoluble inclusion body. In this case, cells 
are collected by centrifugation after cultivation, disrupted by 
ultrasonication, or the like, then subjected to centrifugation, etc. to 
separate the insoluble fraction containing the inclusion body. After being 
washed, the inclusion bodies are solubilized with a commonly used protein 
solubilizer, such as detergent urea or guanidine hydrochloride, followed 
by purification by various chromatographies, such as ion exchange, gel 
filtration, hydrophobic interaction and affinity chromatographies, as 
necessary, after which a refolding treatment by dialysis or dilution is 
conducted to yield a preparation of endoglycoceramidase activator II 
retaining its activity. This preparation may be purified by various 
chromatographies to yield a highly pure preparation of endoglycoceramidase 
activator II. 
Using the endoglycoceramidase activator gene of the present invention, gene 
encoding the desired polypeptide possessing endoglycoceramidase activator 
activity or functionally equivalent variant thereof can be obtained from 
DNAs or cDNAs of other gene sources than that mentioned above by means of 
hybridization. To obtain the desired gene encoding a polypeptide 
possessing endoglycoceramidase activator activity or functionally 
equivalent variant thereof by hybridization, the following method, for 
example, can be used. 
First, chromosomal DNA obtained from the desired gene source, or cDNA 
prepared from mRNA by means of reverse transcriptase, is joined to a 
plasmid or phage vector and introduced into a host to yield a library by a 
conventional method. The library is then cultured on a plate; the 
resulting colonies or plaques are transferred onto a nitrocellulose or 
nylon membrane and subjected to denaturing treatment to immobilize the DNA 
to the membrane. This membrane is incubated in a solution containing a 
probe labeled with .sup.32 P or the like (the probe used may be any gene 
encoding the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, or a 
portion thereof; for example, the genes shown in SEQ ID NO:2 or SEQ ID 
NO:4, or a portion thereof can be used), to form a hybrid between the DNA 
on the membrane and the probe. For example, the membrane with DNA 
immobilized thereon is subjected to hybridization with the probe in a 
solution containing 6 .times. SSC, 1% sodium lauryl sulfate, 100 .mu.g/ml 
salmon sperm DNA and 5 .times. Denhardt's solution (containing bovine 
serum albumin, polyvinylpyrrolidone and Ficoll, each at 0.1%) at 
65.degree. C. for 20 hours. After hybridization, the nonspecifically 
adsorbed portion is washed out, followed by autoradiography, etc. to 
identify clones that formed a hybrid with the probe. This procedure is 
repeated until only a single clone has formed the hybrid. The clone thus 
obtained has a gene encoding the desired protein inserted therein. 
The nucleotide sequence of the gene obtained is then determined by, for 
example, the following method, to confirm if the gene obtained is 
identical with the desired gene encoding a polypeptide possessing 
endoglycoceramidase activator activity or functionally equivalent variant 
thereof. 
When the recombinant is Escherichia coli, nucleotide sequencing for a clone 
obtained by hybridization can be achieved by culturing the Escherichia 
coli in a test tube, or the like, extracting the plasmid by a conventional 
method, digesting the extracted plasmid with restriction enzymes, 
separating the insert and subcloning it into M13 phage vector, or the 
like, and determining the nucleotide sequence by the dideoxy method. When 
the recombinant is a phage, basically the same procedure as that used 
above can be used to determine the nucleotide sequence. Basic experimental 
techniques for from cultivation to nucleotide sequencing are described in, 
for example, Molecular Cloning, A Laboratory Manual, 2nd ed., T. Maniatis 
et al., Cold Spring Harbor Laboratory Press (1989). 
To confirm the identity of the gene obtained as the desired gene encoding a 
polypeptide possessing endoglycoceramidase activator activity or 
functionally equivalent variant thereof, the nucleotide sequence 
determined is compared with the nucleotide sequence of the 
endoglycoceramidase activator gene of the present invention and the amino 
acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 in the sequence listing. 
If the gene obtained does not contain the entire region encoding a 
polypeptide possessing endoglycoceramidase activator activity or 
functionally equivalent variant thereof, the nucleotide sequence of the 
entire region encoding a polypeptide possessing endoglycoceramidase 
activator activity or functionally equivalent variant thereof that 
hybridizes to the endoglycoceramidase activator gene of the present 
invention can be determined by preparing a synthetic DNA primer from the 
gene obtained, and amplifying the lacking region by PCR or by screening 
the DNA library or cDNA library using the gene fragment obtained as a 
probe. 
A polypeptide possessing endoglycoceramidase activator activity or 
functionally equivalent variant thereof can be obtained by gene 
engineering technology as follows: First, the obtained endoglycoceramidase 
activator gene or another gene encoding a polypeptide possessing 
functionally equivalent activity is joined to an expression vector which 
is capable of expressing the gene in an appropriate host cell, such as 
Escherichia coli, Bacillus subtilis, actinomyces, yeast, fungi, animal 
cell, insect cell or plant cell, by a conventional method, followed by 
introduction into the host cell, to yield a recombinant. By culturing this 
recombinant, a polypeptide possessing endoglycoceramidase activator 
activity can be produced. Also, by the use of a cell incapable of sugar 
chain biosynthesis as a host, e.g., a prokaryotic organism, such as 
Escherichia coli and Bacillus subtilis, actinomyces, or by the use of a 
variant yeast, fungi, animal, insect or plant cell which has lost its 
capability of sugar chain biosynthesis, an endoglycoceramidase activator 
polypeptide having no sugar chains can be expressed. 
In some expression systems, the obtained endoglycoceramidase activator gene 
or a gene encoding functionally equivalent variant of endoglycoceramidase 
activator includes a region encoding a signal peptide for cellular 
secretion, resulting in extracellular secretion and accumulation of the 
desired polypeptide in the culture broth. In this case, the desired 
polypeptide can be recovered from the culture broth. When the desired 
polypeptide is accumulated in the recombinant, it may be recovered from 
cultured cells via cell disruption. In addition, when the polypeptide 
expressed in the recombinant is accumulated in the form of an insoluble 
substance (inclusion body), it may be recovered, then solubilized under 
mild conditions, e.g., with urea, followed by denaturant removal, to 
restore the original activity. Expression can be confirmed by determining 
endoglycoceramidase activator activity as described above. 
A polypeptide possessing endoglycoceramidase activator activity can be 
purified from a recombinant by ordinary chromatographic techniques. For 
example, when the desired polypeptide is secreted extracellularly from the 
recombinant, the culture supernatant is subjected to a chromatography, 
such as hydrophobic interaction, ion exchange or gel filtration 
chromatography, to obtain the desired polypeptide as expressed. When the 
desired polypeptide is accumulated in the recombinant, cultured cells are 
disrupted and, if the desired polypeptide is present in a solubilized 
form, the supernatant is subjected to a chromatography, such as 
hydrophobic interaction, ion exchange or gel filtration chromatography, to 
obtain the desired polypeptide as expressed. When the expression product 
is accumulated as an insoluble substance, cells are disrupted, after which 
the precipitate is recovered and solubilized with a denaturant, such as 
urea. The denaturant is then removed, followed by refolding and subsequent 
chromatographic treatment as described above, to obtain a polypeptide with 
desired activity. 
The present invention provides the primary structure of endoglycoceramidase 
activator, and the gene structure thereof. The elucidation of the gene 
structure achieved in the present invention permits the production of a 
polypeptide possessing endoglycoceramidase activator activity by gene 
engineering. By the present method using gene engineering technology, a 
highly pure polypeptide preparation possessing endoglycoceramidase 
activator activity can be produced at low cost. 
EXAMPLES 
The following examples illustrate the present invention but are not 
intended to limit the invention in any manner. 
Example 1. 
Cloning of endoglycoceramidase activator II structural gene 
(1) Extraction and purification of genomic DNA 
Rhodococcus sp. M-777, an endoglycoceramidase activator II producer, was 
inoculated to 30 ml of a medium (pH 7.0) comprising 1.5% mycological 
peptone (produced by OXOID), 0.2% NaCl and 0.1% yeast extract, and 
subjected to shaking culture at 28.degree. C. for 3 days. The culture 
broth was transferred to 900 ml of the same medium and subjected to 
shaking culture at 28.degree. C. for 3 days. After completion of the 
cultivation, the culture broth was centrifuged; cells were collected and 
suspended in 4.5 ml of a buffer (50 mM Tris-HCl, 50 mM EDTA, pH 8.0), then 
frozen and thawed. To this cell suspension, 2.5 ml of a buffer (50 mM 
Tris-HCl, 50 mM EDTA, pH. 8.0) containing 4 mg/ml lysozyme was added, 
followed by incubation at 30.degree. C. for 16 hours. To this mixture, 10 
ml of an extracting buffer (50 mM Tris-HCl, 1% SDS, 0.4 mg/ml proteinase 
K, pH 7.5) was added, followed by incubation at 50.degree. C. for 16 
hours, after which 10 ml of another extracting buffer (50 mM Tris-HCl, 
0.5% SDS, 0.2 mg/ml proteinase K, pH 7.5) was added, followed by 
incubation for 8 hours. After the incubation mixture was allowed to cool 
to room temperature, an equal volume of a phenol/chloroform solution, 
previously saturated with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), 
was added, followed by gentle rotary shaking for 16 hours and subsequent 
centrifugation at 3,500 rpm for 30 minutes, after which the supernatant 
was recovered. This solution was dialyzed against a buffer (10 mM 
Tris-HCl, 5 mM EDTA, pH 8.0) at 4.degree. C. to yield a genomic DNA 
solution. 
(2) Partial amino acid sequencing of endoglycoceramidase activator II 
The 27.9 kDa trypsin digest of endoglycoceramidase activator II was first 
purified by the method described in The Journal of Biochemistry 110, 
328-332 (1991). The N-terminal sequence of the purified 27.9 kDa trypsin 
digest of endoglycoceramidase activator II was then determined by the 
Edman degradation method. As a result, the N-terminal amino acid sequence 
(SEQ ID NO:5) was determined and designated as ACTN. 
The internal amino acid sequence was then determined as follows: 
To a test tube containing 4 .mu.l pyridine, 1 .mu.l 4-vinylpyridine, 1 
.mu.l tributylphosphine and 5 .mu.l distilled water, a smaller sample tube 
containing the purified sample was inserted; the outer tube was vacuum 
sealed, followed by heating at 100.degree. C. for 5 minutes to 
pyridylethylate the cysteine residues in the sample in a gas phase. 
A 1 nmol sample thus S-pyridylethylated was dissolved in 50 .mu.l of a 20 
mM Tris buffer (pH 9.0) containing 4 M urea; 8 pmol of lysylendopeptidase 
(produced by Pierce) was added, followed by digestion at 37.degree. C. for 
16 hours. Reverse-phase chromatography was then conducted on a density 
gradient from distilled water containing 0.065% trifluoroacetic acid (TFA) 
to acetonitrile containing 0.05% TFA through a .mu.RPC C2/C18 SC2.1/10 
column (produced by Pharmacia) using a SMART system (produced by 
Pharmacia), to purify the peptide fragment. The peptide fragment thus 
purified was analyzed by the Edman degradation method using a model 477 
gas-phase peptide sequencer (produced by Applied Biosystems). As a result, 
the internal partial amino acid sequence (SEQ ID NO:6) was determined and 
designated as ACTI. 
(3) Cloning of DNA fragment containing endoglycoceramidase activator II 
gene. 
The genomic DNA prepared in Example 1 (1), 25 .mu.g, was digested with 
restriction enzymes BamHI, PstI and SalI (all produced by Takara Shuzo), 
each 100 U, at 37.degree. C. for 6 hours; after additional 100 U of each 
enzyme was added, the reaction was continued for 16 hours. This reaction 
mixture, in an amount equivalent to 5 .mu.g of DNA, was subjected to 0.7% 
agarose gel electrophoresis, after which the DNA was transferred onto a 
nylon membrane (Hybond-N+, produced by Amersham) by the Southern blotting 
method (Idenshi Kenkyuhou II, pp. 218-221, published by Tokyo Kagaku 
Dojin). This filter was provided in duplicate. 
The hybridization probes used were the oligonucleotides ACTNH (SEQ ID NO:7) 
and ACTIH (SEQ ID NO:8) designed and synthesized on the basis of the 
N-terminal amino acid sequence ACTN (SEQ ID NO:5) and internal partial 
amino acid sequence ACTI (SEQ ID NO:6) determined in Example 1 (2). These 
oligonucleotide sequences were designed on the basis of codons of high 
availability in the host as determined from the known base sequences 
encoding various proteins for the genus Rhodococcus. 
These synthetic oligonucleotides, each 10 pmol, were labeled with .sup.32 P 
using the MEGARABEL.TM. (produced by Takara Shuzo). 
Each of the pair of filters prepared above was subjected to 
prehybridization at 65.degree. C. for 3 hours in a solution containing 6 
.times.SSC (1 .times. SSC is an aqueous solution of 8.77 g of NaCl and 
4.41 g of sodium citrate in 1 l of water), 0.5% SDS, 100 .mu.g/ml herring 
sperm DNA and 5 .times. Denhardt's (containing bovine serum albumin, 
polyvinylpyrrolidone and Ficoll, each at 0.1% concentration), after which 
each of the labeled probes was added to a concentration of 0.5 pmol/ml, 
followed by overnight hybridization at 55.degree. C. Each filter was then 
washed in 6.times. SSC at room temperature for 10 minutes, in 2 .times. 
SSC and 0.1% SDS at room temperature for 10 minutes, and in 0.2.times. SSC 
and 0.1% SDS at 55.degree. C. for 30 minutes, after which excess solution 
was removed; the each filter was exposed to an imaging plate (produced by 
Fuji Photo Film) for 3 hours and the image was detected using a BAS2000 
imaging analyzer (produced by Fuji Photo Film). 
As a result, bands hybridizing to the oligonucleotide ACTNH appeared at 
positions corresponding to about 10 kbp for the BamHI digest, about 2.3 
kbp for the PstI digest, and about 450 bp for the SalI digest. Also, bands 
hybridizing to the oligonucleotide ACTIH appeared at positions 
corresponding to about 10 kbp for the BamHI digest, about 2.3 kbp for the 
PstI digest, and about 1 kbp for the SalI digest. For the subsequent 
experiments, the PstI digest was used, since it is easy to handle. 
The genomic DNA digested with restriction enzyme PstI, 20 .mu.g, was 
subjected to 0.7% agarose gel electrophoresis; a portion corresponding to 
the band appearing in the above-described hybridization was cut out and 
subjected to extraction and purification using the EASYTRAP.TM.(produced 
by Takara Shuzo); the resulting DNA fragment was inserted into the PstI 
site of pUC19 (produced by Takara Shuzo). 
Escherichia coli JM109 was transformed with this plasmid, after which it 
was cultured on 5 round petri dishes 8.5 cm in diameter until 200 to 1,000 
colonies per dish were formed. From these plates, 300 colonies were 
selected and transferred onto a nylon membrane (Hybond-N+, produced by 
Amersham) placed on a plate of medium. After cultivation at 37.degree. C. 
for 3 hours, this nylon membrane was kept on filter paper immersed in a 
solution comprising 0.5 M NaOH and 1.5 M NaCl for 5 minutes (denaturation) 
and on filter paper immersed in a solution comprising a 0.5 M Tris-HCl 
buffer (pH 7.0) and 3 M NaCl for 5 minutes (neutralization), followed by 
rinsing with 2 .times. SSC. Using this nylon membrane and the 
oligonucleotide ACTNH (SEQ ID NO:7) as a probe, hybridization was 
conducted under the same conditions as those described above; three 
positive colonies were obtained. 
These Escherichia coli JM109 transformants were designated as P23, P54 and 
P62, respectively. These transformants were subjected to the alkali lysis 
method to prepare their plasmid DNAs carrying the clones obtained, which 
were designated as pACTP23, pACTP54 and pACTP62, respectively. These were 
analyzed by digestion with several restriction enzymes and gel 
electrophoresis. As a result, these plasmids were found to share the same 
insert. With this finding in mind, pACTP54 was used for the experiments 
that followed. 
Digestion of pACTP54 with restriction enzyme SalI yielded an about 1 kbp 
fragment, about 450 bp fragment, about 300 bp fragment and about 150 bp 
fragment, which were designated as S1, S2, S3 and S4, respectively. These 
fragments were subjected to agarose gel electrophoresis, then extracted 
and purified from the gel, and subcloned into the SalI site of pUC19 
(produced by Takara Shuzo); the resulting plasmids were designated as 
pACTS1, pACTS2, pACTS3 and pACTS4, respectively. These plasmids were 
further digested with appropriate restriction enzymes (SmaI, BalI, NaeI, 
etc.) and subjected to self-ligation using a DNA ligation kit (produced by 
Takara Shuzo) to obtain various deletion variants. The base sequences of 
these deletion variants and pACTP54 were determined from their end by the 
dideoxy method. 
As a result, it was shown that a sequence encoding the N-terminal amino 
acid sequence ACTN, a sequence hybridizing with ACTNH (oligonucleotide 
used as a probe) is present on pACTS2, that a sequence encoding the 
internal amino acid sequence ACTI, a sequence hybridizing with ACTIH 
(oligonucleotide used as a probe) is present on pACTS1, and that the four 
SalI fragments are arranged on pACTP54 in the order of S3, S2, S1 and S4 
(FIG. 1). 
Translation of these nucleotide sequences into amino acid sequences 
demonstrated the presence of a signal-like sequence upstream the amino 
acid sequence of endoglycoceramidase activator II, enabling the deduction 
of the initiation codon. No stop codon was found downstream this frame; 
these sequences were found to encode the 417 amino acid residues (SEQ ID 
NO:9) from the N-terminus of the purified 27.9 kDa trypsin digest of 
endoglycoceramidase activator II. This 417-residue amino acid sequence 
corresponds to a molecular weight of 42 kDa. The base sequence encoding 
this 417-residue amino acid sequence is set forth in SEQ ID NO:10 in the 
sequence listing. pACTP54 was thus proven to contain the sequence encoding 
an N-terminal portion of endoglycoceramidase activator II, which includes 
the 27.9 kDa portion of endoglycoceramidase activator II, the minimum 
essential unit for its activity. 
(4) Cloning of DNA fragment containing the gene encoding C-terminal region 
of endoglycoceramidase activator II 
To cover the full-length endoglycoceramidase activator II gene, a DNA 
fragment encoding the region near the C-terminus, the region which pACTP54 
lacks, was screened for by the Southern hybridization method in the same 
manner as in Example 1 (3). The probe used was the about 0.4 kbp fragment 
obtained by SmaI/PstI digestion of pACTP54, which contains the DNA 
sequence nearest to the C-terminus among the sequences obtained in Example 
1 (3). Specifically, pACTP54 was digested with restriction enzymes SmaI 
and PstI (both produced by Takara Shuzo) and subjected to 1% agarose gel 
electrophoresis; the resulting about 0.4 kbp DNA fragment was cut out. 
This DNA fragment was subjected to extraction and purification using the 
SpinBind.TM. Series II (produced by Takara Shuzo); the resulting purified 
DNA fragment was labeled with .sup.32 P using a BcaBEST.TM. labeling kit 
(produced by Takara Shuzo). The genomic DNA prepared in Example 1 (1), 50 
.mu.g, was digested with restriction enzymes MluI, SacII, ApaI and Eco52I 
(all produced by Takara Shuzo), each 180 U, at 37.degree. C. for 6 hours. 
From this reaction mixture, in an amount equivalent to 10 .mu.g of DNA, 
filters were prepared in the same manner as in Example 1 (3). Each filter 
was subjected to prehybridization at 68.degree. C. for 3 hours in a 
solution containing 6 .times. SSC (1 .times.SSC is an aqueous solution of 
8.77 g of NaCl and 4.41 g of sodium citrate in 1 l of water), 0.5% SDS, 
100 .mu.g/ml herring sperm DNA and 5 .times. Denhardt's (containing bovine 
serum albumin, polyvinylpyrrolidone and Ficoll, each at 0.1% 
concentration), after which the labeled probe was added to a concentration 
of 0.1 pmol/ml for each, followed by overnight hybridization at 68.degree. 
C. 
Each filter was then washed in 6 .times. SSC at room temperature for 10 
minutes, in 2 .times. SSC and 0.1% SDS at room temperature for 10 minutes, 
and in 0.2 .times. SSC and 0.1% SDS at 70.degree. C. for 30 minutes, after 
which excess solution was removed; each filter was exposed to an imaging 
plate (produced by Fuji Photo Film) for 10 minutes and the image was 
detected using a BAS2000 imaging analyzer (produced by Fuji Photo Film). 
As a result, bands hybridizing to the probe appeared at positions 
corresponding to about 4 kbp for the MluI digest, about 1.3 kbp for the 
SacII digest, about 1 kbp for the ApaI digest, and about 0.6 kbp for the 
Eco52I digest. Judging from its size, the Eco52I digest was assumed to 
fail to completely cover the nearly C-terminal region which pACT4 
lacks. With this finding in mind, the digests with restriction enzymes 
Mlul, SacII and ApaI were used in the experiments that followed. 
The genomic DNA digested with restriction enzyme MluI, SacII or ApaI, 20 
.mu.g, was subjected to 0.7% agarose gel electrophoresis; portions 
corresponding to the bands detected in the above-described hybridization 
were cut out and subjected to extraction and purification using the 
EASYTRAP.TM.(produced by Takara Shuzo); the resulting MluI fragment was 
inserted into the MluI site of pUC19M plasmid prepared by inserting and 
ligating a phosphorylated MluI linker (produced by Takara Shuzo) to a 
digest of pUC19 (produced by Takara Shuzo) with restriction enzyme HincII 
(produced by Takara Shuzo) to confer an MluI site to pUC19!; the SacII 
fragment was inserted into the SacII site of pBluescript IISK(-) (produced 
by Stratagene); and the ApaI fragment was inserted into the ApaI site of 
pBluescript IISK(-). 
Escherichia coli HB101 was transformed with these plasmids, after which it 
was cultured overnight on 10 round petri dishes 8.5 cm in diameter 
containing an L-agar medium containing 100 .mu.g/ml ampicillin until 200 
to 500 colonies per dish were formed. From these dishes, 1,000 colonies 
were selected and transferred onto a nylon membrane (Hybond-N+, produced 
by Amersham) placed on a plate of the same medium. After incubation at 
37.degree. C. for 10 hours, this nylon membrane was kept on filter paper 
immersed in a solution comprising 0.5 M NaOH and 1.5 M NaCl for 5 minutes 
(denaturation) and on filter paper immersed in a solution comprising a 0.5 
M Tris-HCl buffer (pH 7.0) and 3 M NaCl for 5 minutes (neutralization), 
followed by rinsing with 2.times. SSC. Using this nylon membrane and the 
about 0.4 kbp DNA fragment obtained by SmaI/PstI digestion of pACTP54 in 
the same manner as above, hybridization was conducted under the same 
conditions as those described above. Two positive signals were obtained 
from colonies containing the ApaI fragment, although no positive signals 
were obtained from colonies containing the MluI or SacII fragment. 
Plasmid DNAs were prepared by the alkali lysis method from the two positive 
colonies and designated as pBAp42 and pBAp69, respectively. These plasmid 
DNAs were digested with several restriction enzymes (ApaI, PstI, SalI, 
Eco52I, BamHI, KpnI, SacI, Eco109I, NaeI, XhoI, SacII, MluI, EcoRI, and 
HindIII) and analyzed with gel electrophoresis. As a result, pBAp42 and 
pBAp69 were found to share the same insert. With this finding in mind, 
pBAp42 was used for the experiments that followed. 
pBAp42 was then digested with several restriction enzymes (PstI, SalI, 
HincII, AccI, and SmaI) and subcloned. 
The nucleotide sequences of these subclones and pBAp42 were determined from 
their end by the dideoxy method, followed by amino acid sequencing using 
synthetic DNA primers synthesized on the basis of the nucleotide sequences 
determined. As a result, the same sequence as that of the about 0.4 kbp 
fragment obtained by SmaI/PstI digestion of pACTP54 was found. 
In addition, a 1,740 bp open reading frame (ORF) was found over the region 
between the PstI fragment in the pACTP54 insert and the ApaI fragment in 
the pBAp42 insert, whose sequence was determined in Example 1 (3). This 
ORF was found to begin at the initiation codon ATG and terminate at the 
stop codon TGA; in the translated amino acid sequence, amino acid 
sequences corresponding to the amino acid sequences ACTN and ACTI obtained 
in Example 1 (2) were found. 
On the basis of the above results, the entire nucleotide sequence and 
primary structure of the endoglycoceramidase activator II gene were 
determined. The results are given in FIG. 1, in which the restriction 
enzyme maps for the pACTP54 and pBAp42 inserts and the positions of these 
inserts and the endoglycoceramidase activator II gene are shown. 
The nucleotide sequence of the ORF of endoglycoceramidase activator II is 
set forth in SEQ ID NO:2 in the sequence listing. The entire amino acid 
sequence of endoglycoceramidase activator II is set forth in SEQ ID NO:1 
in the sequence listing. 
Example 2 
Construction of plasmid for expression of endoglycoceramidase activator II 
A plasmid for endoglycoceramidase activator II expression in Escherichia 
coli was constructed by isolating the endoglycoceramidase II structural 
gene from pACTP54 and pBAp42 as obtained in Example 1, in which the 
segments of the structural gene are separately present, ligating the gene 
to a plasmid appropriate for its expression in Escherichia coli, and 
introducing it to an Escherichia coli cell. 
(1) Construction of plasmid for expression of N-terminal region active 
polypeptide of endoglycoceramidase activator II 
pACTP54 as obtained in Example 1 was digested with restriction enzyme SacII 
and subjected to agarose gel electrophoresis, after which an about 670 bp 
fragment was extracted and purified using the EASYTRAP.TM.(produced by 
Takara Shuzo). After terminal blunting using a DNA blunting kit (produced 
by Takara Shuzo), the fragment was further digested with restriction 
enzyme MluI and subjected to agarose gel electrophoresis, after which an 
about 450 bp DNA fragment was extracted and purified to yield a SacII-MluI 
fragment. 
Next, pACTP54 was digested with restriction enzymes EcoRI and MluI and 
subjected to agarose gel electrophoresis, followed by extraction and 
purification, to yield an about 820 bp MluI-EcoRI fragment. 
These two fragments were inserted into the HincII-EcoRI site of pTV119N 
(produced by Takara Shuzo) using a DNA ligation kit (produced by Takara 
Shuzo). The resulting plasmid, designated as pTAC1, was digested with 
restriction enzyme XbaI, followed by terminal blunting, after which a stop 
linker (SEQ ID NO:11) was inserted. The resulting plasmid was designated 
as pTAC2 (FIG. 2). 
Since this pTAC2 contains a sequence encoding 13 amino acid residues, 
including a lacz.alpha.-derived peptide, upstream the gene encoding 
endoglycoceramidase activator II, it is expected that expression of 
endoglycoceramidase activator II, as a fusion complex with lacz.alpha., is 
induced by use of the SD sequence and initiation codon of lacZ. This pTAC2 
was introduced into the Escherichia coli JM109 strain; after plasmid DNA 
was prepared from the resulting recombinant by the alkali lysis method, 
the base sequence of the insert was identified. The Escherichia coli JM109 
transformed with pTAC2 was designated as Escherichia coli JM109/pTAC2. 
pTAC1 was digested with restriction enzymes HindIII and BamHI, followed by 
terminal blunting and agarose gel electrophoresis, after which an about 
1.3 kbp DNA fragment was extracted and purified. The purified fragment was 
inserted between the HincII site and the blunt-ended NotI site of pET23b 
(produced by Novagen). This plasmid was designated as pEAC001 (FIG. 3). 
Since this pEAC001 contains the T7 .multidot.Tag.TM. sequence (encoding 
amino acid sequence of the first 11 residues of bacteriophage T7 gene 10 
protein, produced by Novagen) and a sequence encoding 14 amino acid 
residues derived from the cloning site, upstream the gene encoding 
endoglycoceramidase activator II, with the His.multidot.Tag.TM. sequence 
(sequence encoding 6 histidine residues, produced by Novagen) downstream 
the endoglycoceramidase activator II gene, it is expected that expression 
of endoglycoceramidase activator II, as a fusion complex of the N-terminal 
417 residues of matured endoglycoceramidase activator II with the 
T7.multidot.Tag.TM. and His.multidot.Tag.TM. is induced. This plasmid was 
introduced into the Escherichia coli JM109 strain; from the resulting 
recombinant, plasmid DNA was prepared by the alkali lysis method, after 
which the nucleotide sequence of the insert was identified. A plasmid 
proven to have been correctly constructed was introduced to Escherichia 
coli BL21(DE3) for expression. The Escherichia coli BL21(DE3) transformed 
with pEAC001 was designated as Escherichia Coli BL21(DE3)/pEAC 001. 
pEAC001 was digested with restriction enzymes BamHI and SphI, followed by 
terminal blunting and self-ligation. The resulting plasmid was designated 
as pEAC101 (FIG. 4). 
This plasmid is expected to inductively express endoglycoceramidase 
activator II, as a fusion complex having a fused protein portion shorter 
than that of pEAC001 by 9 amino acid residues encoded in the sequence 
derived from the cloning site. This plasmid was introduced to the 
Escherichia coli JM109 strain; from the resulting recombinant, plasmid DNA 
was prepared by the alkali lysis method, after which the base sequence of 
the insert was identified. A plasmid proven to have been correctly 
constructed was introduced into Escherichia coli BL21(DE3) for expression. 
The Escherichia coli BL21(DE3) transformed with pEAC101 was designated as 
Escherichia coli BL21(DE3)/pEAC101 deposited under accession number FERM 
BP-5531 at the National Institute of Bioscience and Human-Technology, 
Agency of Industrial Science and Technology; this strain was first 
deposited under the name of Escherichia coli BL2(DE3)/pEAC101 
(registration certificate), but the erroneous designation was corrected as 
above upon issuance of the Microbial Deposit Certificate of Jun. 15, 
1995!. 
pEAC001 was digested with restriction enzyme XhoI, followed by terminal 
blunting, digestion with SmaI and self-ligation, to yield a plasmid 
lacking the sequence encoding 143 residues and retaining the sequence 
encoding 274 N-terminal residues of matured endoglycoceramidase activator 
II of pEAC001. The resulting plasmid was designated as pEAC002 (FIG. 5). 
This plasmid was introduced into the Escherichia coli JM109 strain; from 
the resulting recombinant, plasmid DNA was prepared by the alkali lysis 
method, after which the nucleotide sequence of the insert was identified. 
A plasmid proven to have been correctly constructed was introduced into 
Escherichia coli BL21(DE3) for expression. The Escherichia coli BL21(DE3) 
transformed with pEAC002 was designated as Escherichia coli 
BL21(DE3)/pEAC002. 
(2) Construction of plasmid for expression of endoglycoceramidase activator 
II polypeptide without signal sequence. 
pBAp42 was digested with restriction enzyme ApaI and subjected to agarose 
gel electrophoresis, followed by extraction and purification from the gel, 
to yield an about 1,000 bp BAp42-ApaI fragment. Separately, pEAC101 as 
obtained in Example 2 was digested with restriction enzyme XhoI, followed 
by terminal blunting using a DNA blunting kit (produced by Takara Shuzo); 
to this fragment, a phosphorylated ApaI linker (produced by Takara Shuzo) 
was inserted and ligated, to provide pEAC101 with an ApaI site. This 
plasmid was digested with restriction enzyme ApaI and dephosphorylated 
with Escherichia coli-derived alkaline phosphatase, followed by ligation 
of the BAp42-ApaI fragment, to yield pEAC201 (FIG. 6). 
This plasmid was introduced into the Escherichia coli JM109 strain; from 
the resulting recombinant, plasmid DNA was prepared by the alkali lysis 
method, after which the nucleotide sequence of the insert was identified. 
A plasmid proven to have been correctly constructed was introduced into 
Escherichia coli BL21(DE3) for expression. The Escherichia coli BL21(DE3) 
transformed with pEAC201 was designated as Escherichia coli 
BL21(DE3)/pEAC201. 
Example 3 
Expression of recombinant endoglycoceramidase activator II in Escherichia 
coli 
(1) Expression of polypeptide possessing endoglycoceramidase activator II 
activity in Escherichia coli 
Escherichia coli JM109/pTAC2, Escherichia coli BL21(DE3)/pEAC001, 
Escherichia coli BL21(DE3)/pEAC101, Escherichia coli BL21(DE3)/pEAC002 and 
Escherichia coli BL21(DE3)/pEAC201 as obtained in Example 2 were each 
inoculated to 5 ml of an L medium containing 100 .mu.l/ml ampicillin, and 
subjected to overnight shaking culture at 37.degree. C.; 0.2% of the 
culture broth was further inoculated to 5 ml of the same medium. Upon 
reach of a turbidity (absorbance at 660 nm) of about 0.5 during 
cultivation, IPTG was added to a final concentration of 1 mM, followed by 
shaking culture for 16 hours. After completion of the cultivation, the 
culture broth was centrifuged; cells were collected, suspended in 0.5 ml 
of a 20 mM Tris-HCl buffer (pH 7.9), disrupted by ultrasonication, and 
centrifuged to yield two fractions: an Escherichia coli cell extract as 
the supernatant and a precipitate. The extract and precipitate were 
subjected to SDS polyacrylamide gel electrophoresis and stained with 
Coomassie Brilliant Blue. 
As a result, endoglycoceramidase activator II was not detected in the case 
of Escherichia coli JM 109/pTAC2. On the other hand, a band attributable 
to endoglycoceramidase activator II was detected in the extract and 
precipitate in the case of Escherichia coli BL21(DE3)/pEAC001, Escherichia 
coli BL21(DE3)/pEAC101 and Escherichia coli BL21(DE3)/pEAC201, and in the 
precipitate in the case of Escherichia Coli BL21(DE3)/pEAC002; thereby 
confirming the expression of endoglycoceramidase activator II. Escherichia 
Coli BL21(DE3)/pEAC001 produced the highest concentration of 
endoglycoceramidase activator II in the soluble fraction. Also, the 
expression product of Escherichia coli BL21(DE3)/pEAC201 provided a band 
at a position almost the same as that for the native endoglycoceramidase 
activator II produced by Rhodococcus sp. M-777. 
Next, cell extracts from Escherichia coli BL21(DE3)/pEAC001, Escherichia 
Coli BL21(DE3)/pEAC101 and Escherichia coli BL21(DE3)/pEAC201 were 
digested with trypsin by the method described in the Journal of 
Biochemistry 110, 328-332 (1991). Each digest was subjected to SDS 
polyacrylamide gel electrophoresis, then electroblotted onto a PVDF 
membrane. This membrane was subjected to specific immunological staining 
using an antiserum obtained by immunizing a rat with the 27.9 kDa trypsin 
digest of purified endoglycoceramidase activator II from Rhodococcus sp. 
M-777. As a result, an about 28 kDa band attributable to trypsin digest of 
endoglycoceramidase activator was detected. 
This fact suggests that the endoglycoceramidase activator II produced by 
Escherichia Coli BL21(DE3)/pEAC001, Escherichia coli BL21(DE3)/pEAC101 and 
Escherichia Coli BL21(DE3)/pEAC201 in their soluble fraction occurs as a 
mixture of native structure and miss-folded steric structures. 
(2) Purification of soluble expression product 
Escherichia coli BL21(DE3)/pEAC101 was inoculated to 5 ml of an L medium 
containing 100 .mu.g/ml ampicillin, and subjected to shaking culture at 
37.degree. C. for 16 hours; 0.6 ml of the culture broth was further 
inoculated to 300 ml of the same medium, followed by shaking culture at 
37.degree. C. Upon reach of a turbidity (absorbance at 660 nm) of about 
0.5 in the culture broth, IPTG was added to a final concentration of 1 mM, 
followed by overnight shaking culture at 37.degree. C. 
After completion of the cultivation, cells were collected via 
centrifugation, suspended in 10 ml of a 20 mM Tris-HCl buffer (pH 7.9), 
disrupted by ultrasonication, and centrifuged to separate the supernatant 
from the precipitate. To the supernatant, 10 ml of a 40 mM Tris-HCl buffer 
(pH 7.9) containing 10 mM imidazole and 1 M NaCl was added to yield a 
sample solution. 
This sample solution was subjected to affinity chromatography to purify a 
soluble fraction expression product. Specifically, a column of 
His.multidot.Bind metal chelating resin (produced by Novagen), to which 
Ni.sup.2 + was previously immobilized, was equilibrated with a 20 mM 
Tris-HCl buffer (pH 7.9) containing 5 mM imidazole and 0.5 M NaCl. After 
the sample solution was added to this column, the column was washed with a 
20 mM Tris-HCl buffer (pH 7.9) containing 60 mM imidazole and 0.5 M NaCl, 
followed by elution with a 20 mM Tris-HCl buffer (pH 7.9) containing 1 M 
imidazole and 0.5 M NaCl, to yield 10 ml of a purified soluble expression 
product. 
(3) Refolding and production of recombinant 27.9 kDa endoglycoceramidase 
activator II 
Ten milliliters of the purified soluble expression product from Escherichia 
coli BL21(DE3)/pEAC101 obtained in Example 3 (2) was added drop by drop to 
990 ml of a 20 mM Tris-HCl buffer (pH 7.9) with stirring; the resulting 
dilution was stirred at 5.degree. C. for 4 days to achieve refolding. 
This solution was added to a His.multidot.Bind metal chelating resin 
column, followed by washing and elution in the same manner as above to 
achieve concentration. The buffer for this eluate was replaced with a 25 
mM Tris-HCl buffer (pH 8.0) containing 2 mM CaCl.sub.2 using a Hi-Trap 
desalting column (produced by Pharmacia). After digestion with 2 mg of 
trypsin at 37.degree. C. for 4 hours, additional 2 mg of trypsin was 
added, followed by digestion at 37.degree. C. for 16 hours, to achieve 
conversion to the 27.9 kDa endoglycoceramidase activator II. The digest 
was passed through an anion exchange resin Q cartridge (produced by 
Bio-Rad), previously equilibrated with a 50 mM Tris-HCl buffer (pH 7.9), 
and further passed through an affinity column Protrap.TM.(produced by 
Takara Shuzo) to remove the trypsin, followed by ultrafiltration to obtain 
45 .mu.l of a concentrate. 
Analysis by SDS polyacrylamide gel electrophoresis revealed a single band 
corresponding to a molecular weight of about 28,000. Quantitative 
determination using the BCA.TM. protein assay reagent (produced by Pierce) 
demonstrated a protein content of 11 .mu.g/pl using bovine serum albumin 
as standard. 
Endoglycoceramidase II activating activity was determined by the method 
described in the Journal of Biochemistry 110, 328-332 (1991); 
endoglycoceramidase II activating activity was confirmed. In other words, 
it was proven that about 1.7 mg of the recombinant 27.9 kDa 
endoglycoceramidase activator II is obtained from 1 liter of the culture 
broth of Escherichia coli BL21(DE3)/pEAC101, which includes the gene of 
the present invention. The amino acid sequence of this 27.9 kDa 
endoglycoceramidase activator II is set forth in SEQ ID NO:3 in the 
sequence listing, and its base sequence is set forth in SEQ ID NO:4 in the 
sequence listing. 
Other modifications of the above described embodiments of the invention 
which are obvious to those skilled in the art are intended to be within 
the scope of the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 11 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 580 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
MetSerSerLysLeuTyrArgTyrLeuAlaProValAlaValGly 
151015 
AlaThrValValAlaGlyAlaGlyValLeuGlyValGlyAlaAla 
202530 
SerAlaAlaThrThrIleThrProPheAsnAsnAlaCysGlnAla 
354045 
ThrProSerSerSerLeuAlaGlyGlyProGlnThrGlnValGln 
505560 
AlaAlaSerValThrValAspAlaProGluThrValAlaProGly 
657075 
GluGluPheValValThrIleSerProProProIleSerValPro 
808590 
AsnAspLeuGlySerGlyAlaSerLeuSerAsnIleSerArgLeu 
95100105 
LysIleAspValAlaMetProGluAsnAlaGlnPheIleGlyAla 
110115120 
GluValValAlaGlyThrSerAlaGlyIleThrGlyValAlaPro 
125130135 
AsnValIleValValAsnGluSerGlySerProAspAlaAsnGly 
140145150 
SerIleIleArgLeuSerGlyAsnAsnGluThrIleGlyAsnGly 
155160165 
ProLysSerSerLysSerSerGluGlyGlyIleLysAlaAsnAla 
170175180 
SerGlySerThrThrSerPheGlnLeuProGlnValLysAlaThr 
185190195 
LeuLysAlaGlyAlaAlaGlyGluIleSerMetLysLeuArgThr 
200205210 
AlaGlyAsnAlaGlyGlnPheGlyAsnAspAlaAsnPheLeuThr 
215220225 
PheLeuProArgAlaSerAlaProIleValGlyThrValTrpAla 
230235240 
ProThrGlnCysSerProArgAspThrAlaAlaGlyProLeuAsn 
245250255 
AlaGlyAlaGlyProLeuAlaThrIleGlnIleLeuArgGlnAla 
260265270 
ValAlaThrValSerTyrLeuAspGlyProSerAlaValThrAsn 
275280285 
GlyGlyGluPheThrLeuAsnAlaThrValValProThrProAsp 
290295300 
SerGlyGlnValGlnPheThrArgAspGlyGluAspValGlyAla 
305310315 
ProValAspLeuValAsnGlyLysAlaSerLeuThrGlnSerLeu 
320325330 
AspThrAspGlyAspTyrAlaTyrGluAlaLysPheLeuGlyAla 
335340345 
GluPhePheAsnProSerSerAlaAlaLysThrValThrValThr 
350355360 
SerGlnAspIleGlnThrThrThrSerValThrGlyProAspHis 
365370375 
AspAlaTyrArgAspGlnProValAsnLeuThrAlaLysValGlu 
380385390 
ProGlyValSerGlyGlyThrValAlaPheGluValAspGlyThr 
395400405 
ProValGlyThrAlaAspValMetAspAspGlyAlaAlaValLeu 
410415420 
ProHisThrPheThrThrAsnGlyThrHisArgValIleAlaArg 
425430435 
TyrSerGlyAlaGluGlyIleSerProSerValSerLeuGlnTyr 
440445450 
ProValSerValThrGluAlaProAlaAlaAspValAlaThrThr 
455460465 
IleThrValAspProIleAlaSerThrAlaLysGlySerProVal 
470475480 
ThrLeuThrAlaArgLeuAspProAlaAspAlaArgGlyThrVal 
485490495 
GlnPheLysLeuGlyAspValLeuLeuGlyGlyProValArgVal 
500505510 
AspAlaAsnGlyValAlaThrLeuThrThrPhePheGlnAsnPro 
515520525 
GlyGluPheValValThrAlaGlnPheThrAlaAspAlaGlyPhe 
530535540 
IleAspSerAlaAlaSerProValAsnLeuThrValThrGlyAsp 
545550555 
ProAspThrIleProAsnProGluGlyGlyGlySerLeuAlaGly 
560565570 
LeuSerGlyLeuPheGlySerLeuGlyGly 
575580 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1740 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: genomic DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ATGAGTTCCAAGCTGTACCGCTACCTCGCGCCGGTCGCCGTGGGCGCGACGGTGGTCGCC60 
GGTGCCGGAGTTCTGGGTGTGGGGGCCGCTTCCGCGGCGACGACGATCACGCCGTTCAAC120 
AACGCATGTCAGGCGACGCCGTCGTCGAGCCTGGCCGGTGGGCCACAGACTCAGGTGCAG180 
GCGGCGTCGGTGACGGTCGACGCACCGGAGACCGTCGCTCCCGGTGAGGAGTTCGTGGTG240 
ACGATCTCGCCGCCGCCGATCTCGGTGCCCAACGATCTGGGTTCCGGCGCGAGCCTGTCG300 
AACATCTCGCGGCTCAAGATCGACGTCGCGATGCCGGAGAACGCGCAGTTCATCGGCGCC360 
GAGGTGGTGGCCGGAACGTCAGCGGGCATCACGGGTGTCGCGCCCAACGTCATCGTCGTC420 
AACGAGAGTGGTAGTCCGGACGCGAACGGCTCGATCATCCGGCTGTCCGGAAACAACGAG480 
ACGATCGGCAACGGACCGAAGTCCTCGAAGAGTTCCGAGGGCGGTATCAAGGCGAACGCG540 
TCGGGCAGCACCACGTCCTTCCAGTTGCCGCAGGTCAAGGCCACCCTCAAGGCGGGCGCG600 
GCCGGCGAGATCTCGATGAAGCTGCGCACCGCAGGAAACGCCGGGCAGTTCGGCAACGAC660 
GCGAACTTCCTCACGTTCCTGCCCCGCGCCAGCGCACCGATCGTCGGCACGGTCTGGGCC720 
CCCACCCAGTGCTCGCCGCGTGACACCGCGGCCGGCCCGCTCAACGCGGGAGCCGGTCCG780 
CTGGCCACGATCCAGATCCTGCGGCAGGCGGTCGCCACCGTCAGCTACCTGGACGGTCCG840 
AGCGCGGTGACCAACGGCGGCGAGTTCACGCTCAACGCCACGGTCGTGCCCACCCCGGAC900 
AGCGGTCAGGTCCAGTTCACCCGGGACGGTGAGGACGTCGGCGCGCCGGTCGATCTGGTG960 
AACGGCAAGGCGTCGCTGACGCAGTCGCTCGACACCGACGGCGACTACGCCTACGAGGCG1020 
AAGTTCCTGGGCGCGGAGTTCTTCAATCCGTCGTCCGCCGCGAAGACGGTCACGGTGACC1080 
TCGCAGGACATCCAGACCACCACGTCGGTCACCGGACCTGACCACGACGCCTACCGCGAC1140 
CAGCCGGTGAACCTCACCGCGAAGGTCGAGCCGGGCGTCTCGGGCGGCACGGTGGCTTTC1200 
GAGGTCGACGGAACCCCGGTCGGCACCGCCGATGTGATGGATGACGGCGCGGCGGTGCTC1260 
CCGCACACCTTCACCACCAACGGCACGCACCGCGTGATCGCCCGCTACTCGGGTGCCGAG1320 
GGGATCTCCCCGTCGGTCTCGCTGCAGTACCCGGTCAGCGTCACCGAGGCGCCGGCCGCC1380 
GACGTGGCCACCACGATCACGGTCGATCCGATCGCGTCGACTGCCAAGGGCTCGCCGGTG1440 
ACCCTCACCGCGCGTCTCGATCCGGCCGACGCCCGGGGCACGGTGCAGTTCAAGCTCGGC1500 
GACGTCCTGCTCGGCGGACCGGTGCGGGTGGATGCGAACGGTGTCGCGACACTGACGACG1560 
TTCTTCCAGAACCCCGGCGAGTTCGTCGTCACGGCCCAGTTCACCGCCGACGCCGGTTTC1620 
ATCGACTCGGCAGCGAGCCCGGTTAACCTCACGGTGACCGGTGATCCGGACACCATTCCG1680 
AATCCGGAGGGCGGCGGAAGCCTCGCAGGCCTTTCGGGACTGTTCGGTAGCTTGGGCGGC1740 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 237 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
SerAlaThrThrIleThrProPheAsnAsnAlaCysGlnAlaThr 
151015 
ProSerSerSerLeuAlaGlyGlyProGlnThrGlnValGlnAla 
202530 
AlaSerValThrValAspAlaProGluThrValAlaProGlyGlu 
354045 
GluPheValValThrIleSerProProProIleSerValProAsn 
505560 
AspLeuGlySerGlyAlaSerLeuSerAsnIleSerArgLeuLys 
657075 
IleAspValAlaMetProGluAsnAlaGlnPheIleGlyAlaGlu 
808590 
ValValAlaGlyThrSerAlaGlyIleThrGlyValAlaProAsn 
95100105 
ValIleValValAsnGluSerGlySerProAspAlaAsnGlySer 
110115120 
IleIleArgLeuSerGlyAsnAsnGluThrIleGlyAsnGlyPro 
125130135 
LysSerSerLysSerSerGluGlyGlyIleLysAlaAsnAlaSer 
140145150 
GlySerThrThrSerPheGlnLeuProGlnValLysAlaThrLeu 
155160165 
LysAlaGlyAlaAlaGlyGluIleSerMetLysLeuArgThrAla 
170175180 
GlyAsnAlaGlyGlnPheGlyAsnAspAlaAsnPheLeuThrPhe 
185190195 
LeuProArgAlaSerAlaProIleValGlyThrValTrpAlaPro 
200205210 
ThrGlnCysSerProArgAspThrAlaAlaGlyProLeuAsnAla 
215220225 
GlyAlaGlyProLeuAlaThrIleGlnIleLeuArg 
230235 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 711 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: genomic DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
TCGGCGACGACGATCACGCCGTTCAACAACGCATGTCAGGCGACGCCGTCGTCGAGCCTG60 
GCCGGTGGGCCACAGACTCAGGTGCAGGCGGCGTCGGTGACGGTCGACGCACCGGAGACC120 
GTCGCTCCCGGTGAGGAGTTCGTGGTGACGATCTCGCCGCCGCCGATCTCGGTGCCCAAC180 
GATCTGGGTTCCGGCGCGAGCCTGTCGAACATCTCGCGGCTCAAGATCGACGTCGCGATG240 
CCGGAGAACGCGCAGTTCATCGGCGCCGAGGTGGTGGCCGGAACGTCAGCGGGCATCACG300 
GGTGTCGCGCCCAACGTCATCGTCGTCAACGAGAGTGGTAGTCCGGACGCGAACGGCTCG360 
ATCATCCGGCTGTCCGGAAACAACGAGACGATCGGCAACGGACCGAAGTCCTCGAAGAGT420 
TCCGAGGGCGGTATCAAGGCGAACGCGTCGGGCAGCACCACGTCCTTCCAGTTGCCGCAG480 
GTCAAGGCCACCCTCAAGGCGGGCGCGGCCGGCGAGATCTCGATGAAGCTGCGCACCGCA540 
GGAAACGCCGGGCAGTTCGGCAACGACGCGAACTTCCTCACGTTCCTGCCCCGCGCCAGC600 
GCACCGATCGTCGGCACGGTCTGGGCCCCCACCCAGTGCTCGCCGCGTGACACCGCGGCC660 
GGCCCGCTCAACGCGGGAGCCGGTCCGCTGGCCACGATCCAGATCCTGCGG711 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: N-terminal fragment 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
AlaThrThrIleThrProPheAsnAsnAlaXaaGlnAlaThrPro 
151015 
SerSerXaaLeuAlaGlyGlyProGlnThr 
2025 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(v) FRAGMENT TYPE: internal fragment 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
LeuArgThrAlaGlyAsnAlaGlyGlnPheGlyAsnAspAlaAsn 
151015 
PheLeuThrPheLeuProArgAlaSer 
20 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 44 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GCCACCACCATCACSCCSTTCAACAACGCVCCSCAGGCSACCCC44 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 56 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
ACCGCCGGCAACGCVGGYCAGTTCGGYAACGAYGCVAACTTCCTSACCTTCCTSCC56 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 417 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
AlaThrThrIleThrProPheAsnAsnAlaCysGlnAlaThrPro 
151015 
SerSerSerLeuAlaGlyGlyProGlnThrGlnValGlnAlaAla 
202530 
SerValThrValAspAlaProGluThrValAlaProGlyGluGlu 
354045 
PheValValThrIleSerProProProIleSerValProAsnAsp 
505560 
LeuGlySerGlyAlaSerLeuSerAsnIleSerArgLeuLysIle 
657075 
AspValAlaMetProGluAsnAlaGlnPheIleGlyAlaGluVal 
808590 
ValAlaGlyThrSerAlaGlyIleThrGlyValAlaProAsnVal 
95100105 
IleValValAsnGluSerGlySerProAspAlaAsnGlySerIle 
110115120 
IleArgLeuSerGlyAsnAsnGluThrIleGlyAsnGlyProLys 
125130135 
SerSerLysSerSerGluGlyGlyIleLysAlaAsnAlaSerGly 
140145150 
SerThrThrSerPheGlnLeuProGlnValLysAlaThrLeuLys 
155160165 
AlaGlyAlaAlaGlyGluIleSerMetLysLeuArgThrAlaGly 
170175180 
AsnAlaGlyGlnPheGlyAsnAspAlaAsnPheLeuThrPheLeu 
185190195 
ProArgAlaSerAlaProIleValGlyThrValTrpAlaProThr 
200205210 
GlnCysSerProArgAspThrAlaAlaGlyProLeuAsnAlaGly 
215220225 
AlaGlyProLeuAlaThrIleGlnIleLeuArgGlnAlaValAla 
230235240 
ThrValSerTyrLeuAspGlyProSerAlaValThrAsnGlyGly 
245250255 
GluPheThrLeuAsnAlaThrValValProThrProAspSerGly 
260265270 
GlnValGlnPheThrArgAspGlyGluAspValGlyAlaProVal 
275280285 
AspLeuValAsnGlyLysAlaSerLeuThrGlnSerLeuAspThr 
290295300 
AspGlyAspTyrAlaTyrGluAlaLysPheLeuGlyAlaGluPhe 
305310315 
PheAsnProSerSerAlaAlaLysThrValThrValThrSerGln 
320325330 
AspIleGlnThrThrThrSerValThrGlyProAspHisAspAla 
335340345 
TyrArgAspGlnProValAsnLeuThrAlaLysValGluProGly 
350355360 
ValSerGlyGlyThrValAlaPheGluValAspGlyThrProVal 
365370375 
GlyThrAlaAspValMetAspAspGlyAlaAlaValLeuProHis 
380385390 
ThrPheThrThrAsnGlyThrHisArgValIleAlaArgTyrSer 
395400405 
GlyAlaGluGlyIleSerProSerValSerLeuGln 
410415 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1251 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: genomic DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GCGACGACGATCACGCCGTTCAACAACGCATGTCAGGCGACGCCGTCGTCGAGCCTGGCC60 
GGTGGGCCACAGACTCAGGTGCAGGCGGCGTCGGTGACGGTCGACGCACCGGAGACCGTC120 
GCTCCCGGTGAGGAGTTCGTGGTGACGATCTCGCCGCCGCCGATCTCGGTGCCCAACGAT180 
CTGGGTTCCGGCGCGAGCCTGTCGAACATCTCGCGGCTCAAGATCGACGTCGCGATGCCG240 
GAGAACGCGCAGTTCATCGGCGCCGAGGTGGTGGCCGGAACGTCAGCGGGCATCACGGGT300 
GTCGCGCCCAACGTCATCGTCGTCAACGAGAGTGGTAGTCCGGACGCGAACGGCTCGATC360 
ATCCGGCTGTCCGGAAACAACGAGACGATCGGCAACGGACCGAAGTCCTCGAAGAGTTCC420 
GAGGGCGGTATCAAGGCGAACGCGTCGGGCAGCACCACGTCCTTCCAGTTGCCGCAGGTC480 
AAGGCCACCCTCAAGGCGGGCGCGGCCGGCGAGATCTCGATGAAGCTGCGCACCGCAGGA540 
AACGCCGGGCAGTTCGGCAACGACGCGAACTTCCTCACGTTCCTGCCCCGCGCCAGCGCA600 
CCGATCGTCGGCACGGTCTGGGCCCCCACCCAGTGCTCGCCGCGTGACACCGCGGCCGGC660 
CCGCTCAACGCGGGAGCCGGTCCGCTGGCCACGATCCAGATCCTGCGGCAGGCGGTCGCC720 
ACCGTCAGCTACCTGGACGGTCCGAGCGCGGTGACCAACGGCGGCGAGTTCACGCTCAAC780 
GCCACGGTCGTGCCCACCCCGGACAGCGGTCAGGTCCAGTTCACCCGGGACGGTGAGGAC840 
GTCGGCGCGCCGGTCGATCTGGTGAACGGCAAGGCGTCGCTGACGCAGTCGCTCGACACC900 
GACGGCGACTACGCCTACGAGGCGAAGTTCCTGGGCGCGGAGTTCTTCAATCCGTCGTCC960 
GCCGCGAAGACGGTCACGGTGACCTCGCAGGACATCCAGACCACCACGTCGGTCACCGGA1020 
CCTGACCACGACGCCTACCGCGACCAGCCGGTGAACCTCACCGCGAAGGTCGAGCCGGGC1080 
GTCTCGGGCGGCACGGTGGCTTTCGAGGTCGACGGAACCCCGGTCGGCACCGCCGATGTG1140 
ATGGATGACGGCGCGGCGGTGCTCCCGCACACCTTCACCACCAACGGCACGCACCGCGTG1200 
ATCGCCCGCTACTCGGGTGCCGAGGGGATCTCCCCGTCGGTCTCGCTGCAG1251 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid (synthetic DNA) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
TTAAGTTAACTTAA14 
__________________________________________________________________________