Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO9'/l1378 PCT/DK91/00396
2~87~1
A METEIOD OF CONSTRUCTING SYNTHETIC LEADER SEQUENCES
FIELD OF INVENTION
5 The present invention relates to a method of constructing
synthetic leader peptide sequences for secreting heterologous
polypeptides in yeast, and yeast expression vectors for use in
`; the method.
. ` ~ "
~ lo BACKGROUND OF THE INVENTION
,
` Yeast organisms produae a number of proteins which are
synthesized intracellularly, but which have a function outside
~`~ the cell. Such extracellular proteins are referred to as
lS ~ecreted proteins. These secreted proteins are expressed
initially inside the cell in a precursor or a pre-protein form
containing a presequence ensuring effective direction of the
expressed product across the membrane of the endoplasmic
-` reticulum tER). The presequence, normally named a signal
; 20 peptide, is generalIy cleaved off from the desired product
S`~i during translocation. once entered in the secretory pathway,
the protein is transported to the Golgi apparatus. From the
Golgi the protein can follow different routes that lead to
compartments such as the cell vacuole or the cell membrane, or
25 it can be routed out of the cell to be secreted to the external
` medium (Pfeffer, S.R. and Rothman, J.E. Ann.Rev.Biochem. 56
(1987), 829-852).
. ., ~
Several approaches have been suggested for the expression and
30 secretion in yeast of proteins heterologous to yeast. European
published patent application No. 88 632 describes a process by
which proteins heterologous to yeast are expressed, processed
.~ and secreted by transforming a yeast organism with an ~-
a expression vehicle harbouring DNA encoding the desired protein
35 and a signal peptide, preparing a culture of the transformed
~ organism, growing the culture and recovering the protein from
- the culture medium. The signal peptide may be the signal
-, :
"s
WO~7/ll378 PCT/D~91/00396
;~ 3 ~ ~ 2
peptide of the desired protein itself, a heterologous signal
peptide or a hybrid of native and heterologous signal peptide.
:.,
A problem encountered with the use of signal peptides hetero-
~ 5 logous to yeast might be that the heterologous signal peptide
; does not ensure efficien~ translocation and/or cleavage after
; the signal peptide.
~ The S. cerevisiae MF~1 t~ factor) is synthesized as a prepro
. ~ .
form of 165 amino acids comprising signal-or prepeptide of 19
amino acids followed by a "leader" or propeptide of 64 amino
aicds, encompassing three N-linked glycosylation sites followed
;~ by (LysArg(Asp/Glu, Ala)23~-factor)4 (Kurjan, 3. and
Herskowit2, I. Cell 30 (1982), 933-943). The signal-leader part
of the preproMF~1 has been widely employed to obtain synthesis
~ and secretion of heterologous proteins in S. cerivisiae.
'~
Use of signal/leader peptides homologous to yeast is known from
i.a. US patent specification No. 4,546,082, European published
patent applications Nos. 11~ 201, 123 294, 123 544, 163 529,
;.~ and 123 289 and DK patent application No. 3614/83.
In EP 123 289 utilization of the S. cerevisiae a-factor pre-
cursor is described whereas W0 84/01153 indicates utilization
of the Saccharomyces cerevisiae invertase signal peptide and
~, DX 3614/83 utilization of the Saccharomyces cerevisiae PH05
signal peptide for secretion of foreign proteins.
.. ~
~: US patent specification No. 4,546,082, EP 16 201, 123 294, 123
544, and 163 529 describe processes by which the ~-factor
; signal-leader from SaccharomYces cerevisiae (MF~1 or MF~2) isutilized in the secretion process of expressed heterologous
proteins in yeast. By fusing a DNA sequence encoding the S.
, cerevisiea MF~1 signal/leader sequence at the 5' end of the
3S gene for the desired protein secretion and processing of the
desired protein was demonstrated.
. .
.,
: ,..
''.'.''
..... ...... . . . . .
WOg~/l1318 PCT/~K91/00396
2 ~ n ~ 7~?l1
` EP 206 783 discloses a system for the secretion of polypep~ides
from S. cerevisiae whereby the ~-factor leader sequence has
been truncated to eliminate the four ~-factor peptides present
on the native leader sequence so as to leave the leader peptide
: 5 itself fused to a heterologous polypeptide via the a-factor
~ processing site LysArgGluAlaGluAla. This construction is
-. indicated to lead to an efficient process of smaller peptides
(less than 50 amino acids). For the secretion and procassing
~i of larger polypep~ides, the native ~-factor leader sequence has
~ lO been truncated to leave one or two ~ factor peptides between
`- the leader peptide and the polypeptide.
A number of secreted proteins are routed so as to be exposed
. to a proteolytic processing system which can cleave the peptide
15 bond at the carboxy end of two consecutive basic amino acids.
This enzymatic activity is in S. cerevisiae encoded by the KEX
2 gene (Julius, D.A. et al., Cell 37 (198~b), 1075). Processing
. of the product by the KEX 2 gene product is needed for the
secretion of active S. cerevisiae mating factor ~l (MF~l or ~-
20 factor) but is not involved in the secretion of active S.
cerevisiae mating factor a.
. . .
".;
,' Secretion and correct processing of a polypeptide intended to
'~ be secreted is obtained in some cases when culturing a yeast
:~ 25 organism which is transformed with a vector constructed as
s indicated in the references ~iven above. In many cases, how-
ever, the level of secretion is very low or there is no se-
cretion, or the proteolytic processing may be incorrect or
- incomplete. It is therefore the object of the present invention
30 to provide leader peptides which ensure a more efficient
~, expression and/or processing of heterologous polypeptides.
''''1 .- SUMMARY OF THE INVENTION `~
; - 35 It has surprisingly been found possible to replace the ~-factor
. leader peptide by a variety of different DNA sequences, thereby
obtaining secretion of a heterologous polypeptide in yeast. -
. ;~,
!.
~092/l1378 ?~ u ~ 3 PC~/DK91/00396
sased on this observation, a method has been developed by which
~ random DNA fragments are cloned into yeast vectors downstream
: of a DNA se~uence coding for a signal peptide and upstream of
a DNA sequence coding for a heterologous polypepti~e. A*ter
transformation with the vectors, yeast cells are screened for
secretion of the heterologous polypeptide in question.
~ More specifically, the present invention relates to a method
`.`` of constructing a synthetic leader peptide sequence for
secreting heterologous polypeptides in yeas~, the method
~` comprising
: (a) inserting a random DNA fragment into a yeast expression
~: vector comprising the following sequence
5 1 ~SP~Xn 3 ~ -RS-5 1 ~Xm- (NZT)p-Xq-PS-*gene*-3
.
.: ~
.j wherein SP is a DNA sequence encoding a signal peptide,
. Xn is a DNA sequence encoding n amino acids, wherein n is 0 or
an integer of from 1 to about 10 amino acids,
,~ RS is a restriction endonuclease recognition site for insertion
.~, of random DNA fragments, which site is provided at the junction
.,~
of Xn and Xm,
~ Xm is a DNA sequence encoding m amino acids, wherein m is 0 or
:; 25 an integer from 1 to about 10,
(NZT)p is a DNA sequence encoding Asn-Xaa-Thr, wherein p is 0
::~ or 1,
., .~ .
.. Xq is a DNA sequence encoding q amino acids, wherein q is 0 or
' an integer from 1 to about 10,
PS is a DNA sequence encoding a peptide defining a yeast
processing site, and
*gene* is a DN~ sequence encoding a heterologous polypeptide;
(b) transforming a yeast host cell with the expression vector
of step (a);
.''
-` (c) culturing the transformed host cell of step (b) under
. . 1 , .
.
..,~
WO92/1137~ 2 ~ n ~, rl 3 L PCT/D~91/0~396
appropriate conditi~ns; and
(d) screening the culture of step (c) for secretion of the
heterologous polypeptide.
In the present context, the expression "leader peptide" is
understood to indicate a peptide whose function is to allow the
heterologous polypeptide to be directed from the endoplasmic
reticulum to the Golgi apparatus and further to a secretory ve
sicle for secretion into the medium, (i.e. exportation of the
expressed polypeptide across the cell wall or at least through
the cellular membrane into the periplasmic space of the cell).
The term "synthetic" used in connection with leader peptides
is intended to indicate that the leader peptide constructed by
lS the present method is one not found in nature.
The term "signal peptide" is understood to mean a presequence
.~ ~
which is predominantly hydrophobic in nature and present as an
N-terminal sequence of the precursor form of an extracellular
protein expressed in yeast. The function of the signal peptide
is to allow the heterologous protein to be secreted to enter
~, the endoplasmic reticulum. The signal peptide is normally
~< cleaved off in the course of this process. The signal peptide
# may be heterologous or homologous to the yeast organism
producing the protein but, as explained above, a more efficient
cleavage of the signal peptide may be obtained when it is
'~r homologous to the yeast organism in question.
:. :
The expression "hetero1ogous polypeptide" is intended to
indicate a polypeptide which is not produced by the host yeast
organism in nature. In the method of the invention, the
- heterologous polypeptide is preferably one the secretion of
which by transformed yeast cells may easily be detected, e.g.
by established standard methods such as by immunological
screening by means of antibodies reactive with the polypeptide
in question (cf. for instance Sambrook, Fritsch and Maniatis,
Molecular Cloninq: A Laboratory Manual, Cold Spring Harbor, New ~
., .
: ~.
. .. :, - , . ~,:
WO92~ll378 PCT/DK91/00396
~`York, 1989) or by screening for a specific biological activity
: of the heterologous polypeptide. A positive result of the
screening i~dicates that a leader peptide useful for the
secretion of heterolo~ous polypeptides in yeast has been
constructed.
The expression "a random DNA fragment" is intended to indicate
any se~uence of DNA at least 3 nucleotides in length, for
instance obtained by digesting genomic DNA (of any organism)
~lO with restriction endonuclease(s) or by preparing synthetic DNA,
`~e.g. by the phosphoamidite method described by S.L. Beaucage
and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-
.1869.
. ~.
~15 The peptide Asn-Xaa-Thr encoded hy "(NZT)p" is an asparagine-
".~linked glycosylation site. "Xaa" denotes any one of the known
`~samino acids except Pro.
. . ~ ~;.
In another aspect, the present invention relates to a yeast
expression cloning vector comprising the following sequence
. .
5'-SP-Xn-3'-RS-5'-Xm- (NZT)p-Xq-PS-*gene*-3'
wherein SP, Xn, RS, Xm, (NZT)p, Xq, PS and *gene~ are as defined
2S above.
~ G
~`This vector may be used in the construction of leader peptide
sequences according to the method described above.
In a further aspect, the present invention relates to a yeast
expression vector comprising the following sequence
..~
~5'-SP-Xn-ranDNA-Xm-(NZT)p-Xq-PS-*gene*-3'
. .~ .
35 wherein SP, Xn, Xm, (NZT)p, Xq/ PS and *gene* are as defined ~`
;~above, and ranDNA is a random DNA fragment inserted in a
~-restriction endonuclease recognition site provided at the
,, ~.
.;, .
i ,
WO92/11378 PCT/DK91/00396
2 Q (~ O r~ ~ I
junction of Xn and Xm.
In this vector, the leader peptide sequence (once identified
by the method of the invention) will be composed of the
sequence Xn-ranDNA-Xm-~NZT)p~Xq. Such a vector may be used in
the production of a heterologous polypeptide of interest.
.
In a still further aspect, the present invention relates to a
process for producing a heterologous polypeptide in yeast, the
process comprising culturing a yeast cell, whlch is capable of
expressing a heterologous polypeptide and which is transformed
with a yeast expression vector as described above including a
leader peptide sequence constructed by the method of the
invention, in a suitable medium to obtain expression and
secretion of the heterologous polypeptide, after which the
heterologous polypeptide is recovered from the medium.
DETAILED DISCLOSURE OF THE INVENTION
: . :
;` 20 The length of the random DNA fragment inserted in theexpression vector is not particularly critical. However, in
s order to be of a manageable length, the fragment preferably has
: a length of from 16 to about 600 base pairs. More preferably,the fragment has a length of from about 15 to about 300 base
/ 25 pairs. It is at present considered that a suitable length of
-~ the fragment is from about 30 to about 150 base pairs.
... .
- The random DNA fragment preferahly encodes a high proportion
- of polar amino acids. These are selected from the group
consisting of Glu, Asp, Lys, Arg, His, Thr, Ser, Asn and Gln.
-, In the present context, the term "a high proportion of" is
understood to indicate that the DNA fragment encodes a larger
number of polar amino acids than do other DNA sequences of a
corresponding length. Independently hereof, or in addition
hereto, it may be advantageous that the fragment encodes at
least one proline.
, .i~
' ~' .
. s
.,~
. ' ' , :
WO92tl1378 PCT/D~91/00396
~ 1~ 3 ~
In the sequence 5'-SP-Xn-3'-RS-5'-Xm-(NZT)p-Xq-PS-*gene*-3', n
and/or m and/or q are preferably >1. In particular, all of n,
m and q are ~1.
There is some evidence (cf. WO 89~02463) to support that the
presence of an asparagine-linked glycosylation site in the
leader sequence may confer a higher secretion efficiency to the
leader peptide. In the sequence 5l-SP-Xn-3'-RS-5'-Xm- (NZT) p~Xq~
PS-*gene~-3', p is therefore preferably 1.
The signal peptide sequence (SP) may encode any signal peptide
~which ensures an effective direction of the expressed
- heterologous polypeptide into the secretory pathway of the
cell. The signal peptide may be a naturally occurring signal
`15 pep~ide or funccional parts thereof, or it may be a synthetic
~` peptide. Suitakle signal peptides have been found to be the ~-
factor signal peptide, the signal peptide of mouse salivary
amylase, a modified carboxypeptidase signal peptide, the yeast
BAR1 signal peptide or the Humicola lanuqinosa lipase signal
peptide, or a derivative thereof. The mouse salivary amylase
,signal sequence is described by O. Hagenbuchle et al., Nature
~289, 1981, pp. 643-646. The carboxypeptidase signal sequence
is described by L.A. Valls et al., Cell 48, 1987, pp. 887-897.
lThe BAR1 signal peptide is disclosed in WO 87/02670. The H.
lanu~inosa lipase signal peptide is disclosed in EP 305 216.
,
The yeast processing site encoded by the DNA sequence PS may
suitably be any paired combination of Lys and Arg, such as Lys-
Arg, Arg-Lys, Lys-Lys or Arg-Arg, which permits processing of
the heterologous polypeptide by the KEX2 protease of
- Saccharomvces cerevisiae or the equivalent protease in other
^ yeast species (D.A. Julius et al., Cell 37, 1984, 1075 ff.).
~'If KEX2 processing is not convenient, e.g. if it would lead to
cleavage of the polypeptide product, a processing site for
'35 another protease may be selected instead comprising an amino
acid combination which is not found in the polypeptide product,
e.g. the processing site for FXa, Ile-Glu-Gly-Arg (cf.
, ~ .
:.
:
WO92/1137X PCT/DK91/0~396
?~ t3 ~
. ` ,
Sambro~k, F~i~sch and Maniatis, Molecular Cloninq: A Laboratory
Manual, Cold Spring Harbor, New York, 1989).
,
The heterolo~ous protein produced by the method of the inven-
- 5 tion may be any protein which may advantayeously be produced
;in yeast. Examples of such proteins are aprotinin, tissue
factor pathway inhibitor or other protease inhibitors, insulin
or insulin precursors, human or bovine growth hormone,
interleukin, glucagon, tissue plasminogen activator,
transforming growth factor ~ or ~, platelet-derived growth
` factor, enzymes, or a functional analoyue thereof. In the
present context, the term "functional analogue" is meant to
indicate a polypeptide with a similar function as ~he native
protein (this is intended to be understood as relating to the
,~A 15 nature rather than the level of biological activity of the
native protein). The polypeptide may be structurally similar
` to the native protein and may be derived from the native
protein by addition of one or more amino acids to either or
both the C- and N-terminal end of the native protein,
substitution of one or more amino acids at one or a number of
different sites in the native amino acid sequence, deletion of
one or more amino acids at either or both ends of the native
protein or at one or several sites in the amino acid sequence,
or insertion of one or more amino acids at one or more sites
-~ 25 in the native amino acid sequence. Such modifications are well
. . ., :
~known for several of the proteins mentioned above. ~
.. .~ . .:
"";JThe random DNA fragment and the sequence 5 ~-SP-Xn-31-RS-51~Xm~
6(NZT)p-Xq-PS-*gene* 3' may be prepared synthetically by
30 established standard methods, e.g. the phosphoamidite method
described by S.L. Beaucage and M.H. Caruthers, Tetrahedron
Letters 22, 1981~ pp. 1859-1869~ or the method described by
Matthes et al., EMBO Journal 3 ~ 1984 ~ pp. 801-805. According
to the phosphoamidite method, oligonucleotides are synthèsized,
35 e.g. in an automatic DNA synthesizer, purified, annealed,
ligated and cloned into the yeast expression vector. It should
be noted that the sequence 5'~SP~Xn~3'~RS~5'~Xm~(NZT)p~Xq~PS~
.,.'
. .
',",'~
.,
: .: .: . .. . ~ ,: ,, ::: : .: : : : , -
WO9~/ll37X PCT/DK91/00396
~ iu~ 10
*gen~*-3' need n~t be prepared in a single operation, but may
b~ assembled from ~wo or more oligonucleotides prepared
synthetically in this fashion.
The random DNA fragment or one or more parts of the sequence
5'-SP-Xn-3'-RS-5'-Xm- (NZT)p-Xq-PS-*gene*-3' may also be of
genomic or cDNA origin, for instance obtained by preparing a
-` genomic or cDNA library and screening for DNA sequences coding
for said parts (typically SP or *gene*) by hybridization using
synthetic oligonucleotide probes in accordance with standard
techniques (cf. Sambrook, Fritsch and Maniatis, Molecular Clon-
inq: A Laboratorv Manual, Cold Spring Harbor, New York, 1989).
In this case, a genomic or cDNA sequence encoding a signal
: peptide may be joined to a genomic or cDNA sequence encoding
`~ 15 the heterologous protein, a~ter which the DNA sequence may be
modified by the insertion of synthetic oligonucleotides
encoding the sequence Xn~3'~RS~5l~Xm~~NZT)p~Xq~PS in accordance
~- with well-known procedures.
! l
Finally, the random DNA fragment and/ or the sequence 5'-SP-X~-
3'-RS-5'-Xm-(NZT)p-Xq-PS-*gene*-3' may be of mixed synthetic and
genomic, mixed synthetic and cDNA or mixed genomic and cDNA
origin prepared by annealing fragments of synthetic, genomic
~`~ or cDNA origin (as appropriate), the fragments corresponding
to various parts of the entire DNA sequence, in accordance with
~-~ standard techniques. Thus, it may be envisaged that the DNA
sequence encoding the signal peptide or the heterologous
~; polypeptide may be of genomic or cDNA origin, while the
~ sequence Xn-3'-RS-5'-Xm-(N~T) -X -PS may be prepared
;~ 30 synthetically.
.~ `,!
Preferred DNA constructs encoding insulin precursors are as
shown in Sequence Listings ID Nos. 1-13, or suitable modifica-
`, tions thereof. Examples of suitable modifications of the DNA-3 35 sequence are nucleotide substitutions which do not give rise
to another amino acid sequence of the protein, but which may
correspond to the codon usage of the yeast organism into which
,. ~'' ' :
:
` r
'.. .~ ' ~ . ~ :
WO9Z/I1378 PCT/D~91/00396
~, n ~) ,,(,~ 7 3 1
11
the DNA construct is inserted or nucleotide substitutions which
do give rise to a different amino acid sequence and therefore,
possibly, a different protein structure~ Other examples of
possible modifications are insertion of three ar multiples of
three nucleotides into the sequence, addition of three or
multiples of three nucleotides at either end of the sequence
and deletion of three or multiples of three nucleotides at
either end of or within the sequence.
The recombinant expression vector carrying the sequence 5'-SP-
Xn-3'-RS-5'-Xm-(NZT)p-Xq-ps-*gene*-3~ or 5'-SP-Xn-ranDNA-Xm-
(NZT)p-Xq-PS-*gene*-3' may be any vector which is capable of
replicating in yeast organisms. In the vector, either DNA
sequence should be operably connected to a suitable promoter
sequence. The promoter may be any DNA sequence which shows
transcriptional activity in yeast and may be derived from genes
encoding proteins either homologous or heterologous to yeast.
.,.'! The promoter is preferably derived from a gene encoding a
~, protein homologous to yeast. Examples of suitable promoters are
~ 20 the Saccharomyces cerevisiae MF~l, TPI, ADH or PGK promoters. ~
.,.~ .;.
i~` The sequences shown above should also be operably connected to
' a suitable terminator, e.g. the TPI terminator (cf. T. Alber
: and G. Kawasaki, J._Mol. Ap~l. Gene*. 1, 1982, pp. 419-434).
The recombinant expression vector of the invention further
;. comprises a DNA sequence enabling the vector to replicate in
yeast. Examples of such sequences are the yeast plasmid 2~
~:i replication genes REP 1-3 and origin of replication. The vector
may also comprise a selectable marker, e.g. the Schizo-
:
saccharomvces pombe TPI gene as described by P.R. Russell, Gene
40, 1985, pp. 125-130.
The procedures used to ligate the sequence 5'-SP-Xn-3'-RS-5'-
Xm-(NZT)p-Xq-PS-*gene*-3', the random DNA fragment, the promoter
and the terminator, respectively, and to insert them into
suitable yeast vectors containing the information necessary for
.. .. .
:, .
~:
WOg2/ll378 PCT/DK9t/00396
S ` 12
.
`~ yeast replication, are well known to persons skilled in the art
(cf., for instance, Sambrook, Fritsch and Maniakis, oP~cit.).
It will be understood that the vector may be constructed either
by first preparing a DNA construct containing the entire
- 5 sequence 5 1 -SP-Xn-3 1 -RS-5 1 ~Xm~ (NZT) p-Xq-PS-*gene*-3~ and
subsequently inserting this fragment into a suitable expression
vector, or by sequentially inserting DNA fragments containing
genetic information for the individual elements (such as the
signal peptide, the sequence Xn-3'-RS-5'-Xm- (NZT) p~Xq or the
lo heterologous polypeptide) follow2d by ligation.
''
The yeast organism used in the method of the invention may be
any suitable yeast organism which, on cultivation, produces
large amounts of the heterologous polypeptide in question.
15 Examples of suitable yeast organisms may be strains of the
yeast species Saccharomyces cerevisiae, Sacch _ mYces kluyverl,
Schizosaccharomvces Pombe or Saccharomvces uvarum. The
transformation of the yeast cells may for instance be effected
~ by protoplast formation followed by transformation in a manner
S,~ 20 known per se. The medium used to cultivate the cells may be any
conventional medium suitable for growing yeast organisms. The
secreted heterologous protein, a significant proportion of
which will be present in the medium in correctly processed
form, may be recovered from the medium by conventional pro-
? 2~ cedures including separating the yeast cells ~rom the medium
~-' by centrifugation or filtration, precipitating the protein-
.~, aceous components of the supernatant or filtrate by means of
`. a salt, e.g. ammonium sulphate, followed by purification by a
variety of chromatographic procedures, e.g. ion exchange
30 chromatography, affinity chromatography, or the like.
:, -
BRIEF DESCRIPTION OF THE DRAWINGS
. :
The present invention is further illustrated with reference to
~ 35 the appended drawings wherein
;~ Fig. 1 schematically shows the construction of pMT742~; -
.:
,: -"
.:
, .:
WO92/ll378 PCT/DK91/00396
2 0 9 . ~ 3
Fig. 2 schematically shows the construction of pLaC202;
,
Fig. 3 shows the DNA sequence and derived amino acid sequence
at the cloning site in pLaC202 for random DNA fragments (it
should be noted that the sequence is cleaved in the unique ClaI
site and that ligation without insertion of r~ndom DN~ will
lead to a change in the reading frame);
Fig. 4 schematically shows the construction of pLSC6315DX;
: 10
The invention is further described in the following examples
which are not to be construed as limiting the scope of the
invention as claimedO
..
; EXAMPLES
``. ` ;':
; Plasmids and DNA materials
All expression plasmids are of the C-POT type. Such plasmids
are described in EP patent application No. 171 142 and are
characterized in containing the SchizosaccharomYces pombe
triose phosphate isomerase gene (POT) for the purpose of
plasmid selection and stabilization. A plasmid containing the
POT-gene is available from a deposited E. coli strain (ATCC
39685)~ The plasmids furthermore contain the S. cerevisiae
~ triose phosphate isomerase promoter and terminator (PTp~and
., TTPI). They are identical to pMT742 (M. Egel-Mitani et al., Gene
73, 1988, pp. 113-120) (see fig. 1) except for the region
defined by the Sph-XbaI restriction sites encompassing the PTPI
and the coding region for signal/leader/product.
' The PTPI has been modified with respect to the sequence found
in pMT742, only in order to facilitate construction work. An
internal SphI restriction site has been eliminated by SphI
cleavage, removel of single stranded tails and religation.
~ Furthermore, DNA sequences, upstream to and without any impact
'', -
WO92/ll378 PCT/DK91/00396
~rl~ ~ 14
; on the promoter, have be~n ramoved by Bal3~ exonuclease
treatment followed by ad~i~ion of an SphI restriction site
linker. This promoter construction present on a 373 bp SphI-
EcoRI fragment is designated PTPI~ and when used in plasmids
already described this promoter modification is indicated by
the addition of a ~ to the plasmid name, e.g. pMT742~ (fig. 1).
`:~
`` The assembly of various DNA fragments have occasionally taken
place in a smaller E. coli plasmid of the pT7 type previously
10 described ~cf. W0 89/02463) only modified with respect to the
PTPI as described above, i.e. pT7~. For random cloning described
; below, genomic DNA of various origins have been employed. S.
cerevisiae DNA was isolated ~rom strain MT633 (deposited on 7
December 1990 in the ~eutsche Sammlung von Mikroorganismen und
Zellkulturen under the provisions of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for
the Purpose of Patent Procedure with the deposit number DSM
6278). A. oryzae DNA was isolated from strain A1560 (IF0 4177).
,.;;,.
`~i 20
-.,
Finally a number of synthetic DNA fragments have been employed
~:~ all of which were synthesized on an automatic DNA synthesizer
i (Applied Biosystems model 380A) using phosphoramidite chemistry
and commercially available reagents (S.L. Beaucage and M.H.
25 Caruthers (1981) Tetrahedron Letters 22, 1859-1869). The
oligonucleotides were- purified by polyacrylamide gel
`~ electrophoresis under denaturing conditions. Prior to annealing
~ complementary pairs of such DNA single strands these were
;i kinased by T4 polynucleotide kinase and ATP.
`^ 30
All other methods and materials used are common state of the
art knowledge (J. Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press) Cold
Spring Harbor, N.Y. 1989).
` 35
.... .
,
: j :
,, ~ .
:
....
WOg~ 378 PCT/DK91/0039fi
~ o n -3 ~J ~
xample 1
Construction of PLac2o2
The 490 bp SphI-ApaI of pT7 196~ ~cf. WO 89/02463, fig. 5) and
the 179 bp HinfI-XbaI fragment of pT7.~MI3 (cf. WO 89/02~63,
fig. 1) joined in the 11 kb XbaI-SphI fragment of pMT742 via
; the synthetic adaptor: ;
":
NOR367/373: CAACCATCGATAACACCACTTTGGCTAAGAG
CCGGGTTGGTAGCTATTGTGGTGAAACCGATTCTCTAA
'::
resulting in the plasmid pLaC202 (fig. 2 and 3 as well as
Sequence Listing ID No. 1).
This vector containing a unique ClaI site constitutes one
embodiment of the random DNA cloning vector in which the
`~ product gene codes for the insulin precursor MI3 (B(1-29)-Ala-
~ Ala-Lys-A(1-21)). The following examples concerns the leaders
`~ 20 cloned via this construct.
`~i
ExamPle 2
. ...
: ..,
i 25 Construction of ~LSC6315 and pLSC5210
"... ~ .
Total DNA was isolated from S. cerevisiae strain MT663, and
digested by TaqI, HinPI or TaqI + HinP I. The digests were
separated according to size on a 1% agarose gel, and fragments
` 30 smaller than 600 bp were isolated from each of the three
. digestions.
:~
.,
pLaC202, previously digested with ClaI, prevented from self
ligation with Calf Intestine Alkaline Phosphatase (CIAP),
dephosphorylation, was mixed with the fragment pools described
above and ligated. E. coli strain MT172 (MT172 = MC 1000 m r~
ara~ leuB-6; MC 1000 (cf. M. Casadaban and S. Cohen,
., p
: .. .
~. - .
~092~l137~ ~ PCTtDK91/003g6
~S~I~?J ~
16
J.Mol.Biol. 138, 1980, p. 179)) was transformed with above
ligation mixture, and appr. S000 ApR transformants for each
mixture were o~tained. Recombinant plasmids were prepared from
each of the three types in pools encompassing all 5000
transformants. These plasmid pools were used to transform S.
cerevisiae strain MT663 and the resulting TPI transformants
were immunoscreened for MI3 secretion.
Among the surprisingly large number of posltive transformants
the eight apparently most efficient w~re reisolated and the
plasmid content isolated therefrom.
:
As a result of ~his procedure, it is expected that most of the
- yeast transformant obtalned have a heterogeneous population of
plasmids and to obtain true clones, a step of plasmid
reisolation was therefore performed. The plasmid preparations
from each of the eight reisolated yeast transformants were used
to transform E. coli strain MTl72 to ApR. Plasmids from 12 E.
~`- coli transformants for each of the eight yeast isolates, were
individually used to transform yeast strain MT663, TPI, and MI3
secreting transformants were identified by immunoscreening.
~ Sequencing of the inserts of the eight isolated pLaC202
; derivatives showed three different sequences, two of which,
: - 25 pLSC6315 and pLSC5210, most efficiently support MI3 secretion.
The sequences of the cloned DNA and flanking regions are shown
in Sequence Listings ID Nos. 2 and 4, respectively.
: ,;
: :-~
;- 30 Exam~le 3
. . .
, ......
.~ Modifications of PLSC6315
::
;:~
~' pL5C6315 was chosen for further modification of the cloned
35 synthetic leader sequence.
'"'
~. pLSC6315 was digested with the ApaI endonuclease followed by
.-,!
.:,-' .
:-,:,:
:i
... .
WO9~11378 PCT/DK91/00396
20n~7,~.1
treatment with the exonuclease Bal31. After phenol extraction
the resulting DNA was digested with XbaI and DNA fragments
smaller than the original 367 bp ApaI-XhaI fragment, were
isolated.
pLaC202 was digested with ClaI, and the single stranded CG
tails generated were removed, followed by XbaI digestlon and
" isolation of the 11 Kb XbaI-]ClaI~ fragment ("] [" indicates
; that the single-stranded t~ils have been trimmed off). This
fragment was mixed with the pLSC6415 fragments isolated above
and ligated (fig. 6).
The transformation and screening procedure described in example
2 was repeated, and pLSC6315D3 and pLSC6315D7 were isolated as
plasmids supporting MI3 secretion more efficiently than the
original pLSC6315 (cf. Sequence Listings ID Nos. 6 and 8,
respectively).
.:!
.~....
Exam~le 4
: . ~ Construction of PLAOl - 5
:,,
Total DNA from AsPerqillus or~zae strain A1560 was treated as
- 25 previously described for S. cerevisiae DNA in example 2, and
..... ~ .
; cloning and recloning was performed exactly as described in
. example 2, except that the number of E. coli transformants in
the first cloning was reduced to approximately 3000 per
:J, ligation mixture. This experiment resulted in the isolation of
` 30 five clones of A. orvzae DNA which in the pLaC202 context
mediates secretion of the insulin precursor MI3, from S.
. . .
,!, cerevl~slae.
Sequencing of the inserts showed 5 different sequences, two of -~
which (pLAO2 and pLAO5) are more efficient where MI3 secretion
is concerned. The sequences of the DNA inserts in pLAO2 and
~ pLAO5 are shown, together with the flanking regions, in
',A.
'' :
"s
. ; .. . . . . . . .. . . . . . . . .
WO92/ll37X PCT/DK91/00396
~rv~3s~ 3 ~ 18
sequence Listings ID Nos. lo and 12, respectively.
`: :
Example 5
:` :
Yeast strains harbouring plasmids as described above, were
grown in YPD medium (Sherman, F. et al., Methods in Yeas~
Genetics, Cold Spring Harbor Laboratory 1981). For each strain
6 individual 5 ml cultures were shaken at 30C for 60 hours,
with a final OD600 of approx. 15. After centrifugation the
supernatant was removed for HPLC analysis by which method the
concentration of secreted insulin precursor was measured by a
method described by Leo Snel et al. (1987) Chromatographia 24,
~ 329-332.
.,
In table I the expression levels of insulin precursor, MI3, by
~` use of leader sequences isolated according to the present
-` invention, are given as a percentage of the level obtained with
~ transformants of pMT7~2~, utilizing the MF~(1) leader o~ S.
'`:'iJ cerevisiae.
` Table I
pMI'742 100~
";;Ji 25 pLSC6315 100%
pLSC5210 60%
pLSC6315D3 175%
pLSC6315D7 120%
pLA02 120%
~` 30 pLAO5 60%
:
., ~
: .
.
''- .
:.
r
Wo 92/1~378 PC~/DK~1/00396
2 ~ J ~ 7 3 ~
19
SEQUENCE LISTING
( 1 ) GENERAL INFORMATION:
` (i) APPLICANT: Novo Nordisk A/S
(ii) TITLE OF INVENTION: A Method of Constructing Synthetic
Leader Sequences
(iii) NUMBER OF SEQUENCES: 13
-: ( iv) CORF~ESPONDENCE ADDRESS:
(A) ADDRESSEE: Novo Nordisk A/S, Patent Department
; (B) STREET: Novo Alle
~ (C) CITY: Bagsvaerd
:~` (E) COUNTRY: Denmark
(F) ZIP: DK-2880
(v) COMPUTER READABLE FORM:
. (A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Releas2 #1.0, Version #1.25
:. (vi) CURRENT APPLICATION DATA: ~-
-c~ (A) APPLICATION NUMBER:
. (B) FILING DATE: -
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATXON:
:~i (A) NAME: Thalsoe-Madsen, Birgit
(C) REFERENCE/DOCKET NUMBER: 3540.204-WO
., .. ~. . .
i.~. (ix) TELECOMMUNICATION INFORMATION~
.~ (A) TELEPHONE: +45 4444 8888
(B) TELEFAX: +45 4449 3256
(C) TELEX: 37304
, ' ,
.~
' (2) INFORMATION FOR SEQ ID NO:l:
. .
(i) SEQUENCE CHARACTERISTICS:
~' (A) LENGTH: 335 base pairs
(B) TYPE: nucleic acid
:. (C) STRANDEDNESS: single
. (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
,
. ,j ,
: (xi) SEQUENCE DESCRIPTqON: SEQ ID NO:l: ~:
~i Ga~FKY~I~ AA~ T CA~ACA~GAA GATTAC~AAC TATCAATITC AIACAC~ATA 60
. ~ .
TA~AC~ATTA A~AT&AA AG~CFKX~G C~ CC TC~ATT CD3C~ 120
. ~ . . .
7~
WO 92/11378 PCr/VK91/00396
~ 9~3~ 20
C~AC~ ~ACACCPCT TI~GCrAAGA GAl~GrrAA CC~ACACr~ ~ 180
` ~ AC~A A~AC T~TI~ ~AGG TII~AC AC~G 240
c~crhAGGG ~7sOE~A C~5~R CC~c~ C~I~AC C~A 300
ACrACIr;CPA CrA~CGCA~ r~ 335
. :. -.
( 2 ) INFORM~ION FOR SE)Q ID N0: 2:
(i~ SEX2~TCE ~RACrERIST~CS:
(A) LE:~: 492 base pairs
(B) TY~: nucleic acid
(C) SrRANDElX~ESS: single
(D) ~POL~: linear
'' (ix) ~E:
~ (A) NP~/~CEY: CDS
: (B) ~)CAlICN: 76... 468
: ~ (ix) P~:
(A) N~ME/XEY: sig_peptide
: (B) LOCATION: 76... 309
(ix) ~æ:
(A) NAME/KEY: mat_peptide
~: (B) LOC~IION: 310.. .468
. (xi) SEQUENCE DESCRIPqION: SEQ ID N0:2:
,
:~ GPAITCaIrC AAGAA~AGTT CAAACAAGAA GAIIACAA~C ~C~AIrTC AIACACAATA 60
:
:~ IAAACGATIA A~A~A AIG AAA GTC TTC CTG CTG CrT TCC crc ATT GGA TTC lll
~`............................. Met ~ys Val Phe Leu Leu Leu Ser Leu Ile Gly Ph~
:- -78 -75 -70
;~ TGC TGG GCC CAA CCA TCG A~A GAT GGA ACA CAT TTT CCG AAC AAC AAT 159
~ Cys Trp Ala Gln Pr~ Ser Ile Asp Gly Thr His Phe Pro Asn Asn Asn
; -65 -60 -5S
GTC CCA AXA GAC ACA AGA AAA GAA GGA CTA CAG CAT GAT TAC GAT ACA 207
Val Pro Ile Asp Thr Arg Lys Glu Gly Leu Gln ~is Asp Tyr Asp Thr
. -50 -45 -40 -35
:::
GAA ATT TTG GAG CAC AIT GGA AGC GAT GAG TTA AIT TIG AAT GAA GAG 255
. Glu Ile Leu Glu His Ile Gly Ser Asp Glu Leu Ile Leu Asn Glu Glu
~~ -30 -25 -20
.: lAr GTT AIT GAA AGA ACT TTG CAA GCC ATC GA~ AAC ACC ACT TIG GCT 303
Tyr Val Ile Glu Arg Ihr Leu Gln Ala Ile Asp Asn Thr Thr Leu Ala
. -15 -10 -5
AAG AG~ TTC GTT AAC CAA CAC TTG TGC GGT TCC CAC TTG GTT GAA GCT 351
:~ Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser His TPU Val Glu Ala
,, .
.,
:, .
: .. ~.. -.. . - . .
. ~ . . ~ . ~ . . . . .. .
W O 92/1137X PCT/DK91/00396
2 ~ ~ S 7~ 1
TTG TAC TTG GIr TGC GGT GP~ AG~ GGT TTC TTC IAC AC~ CCT AAG GCT 399
Leu Tyr keu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ala
15 20 25 30
GCT A~ GGT ATT GTC GAA CAA TGC TGT ACC TCC ATC TGC TCC TTG TAC 447
Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr
35 40 45
CAA TTG GAA AAC TAC TGC AAC TAG~CGCAGC CY3C~EGCTC TAGA 492
:~ Gln Leu Glu Asn Tyr Cys Asn
' 50
.
: (2) INFO~M~TqON FOR SEQ ID NO:3,
.~
(i) SEQUEN OE CH~RACTERISl~lCS:
-~ (A) LÆNGTH: 131 am m o acids
(B) TYPE: amlno acid ~.
.~ (D) TOPOLCGY: linear
(ii) MOLECULE TYPE: protein
(xi) SE2UENCE DESCRIPTION: SEQ ID NO:3:
. Met Lys Val Phe Leu Leu Leu Ser Leu Ile Gly Phe Cys Trp Ala Gln
. -78 -75 -70 -65
.,:'!, Pro Ser Ile Asp Gly Thr His Phe Pro Asn Asn Asn Val Pro Ile Asp
, -60 -55 ~50
`~ m r Arg Lys Glu Gly Leu Gln His Asp Iyr Asp Thr Glu Ile Leu Glu
~40 35
. His Ile Gly Ser Asp Glu Leu Ile Leu Asn Glu Glu Tyr Val Ile Glu ~
.$ -30 -25 -20 -15 - .
:~ Arg Thr Leu Gln Ala Ile Asp Asn Thr Thr Leu Ala Lys Arg Phe Val
~ 10 -5
: r,
, Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val
'
:;. Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ala Ala Lys Gly Ile
.. 20 25 30
., Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn
' 35 40 45 50
Tyr Cys Asn
~ .
(2) INFORM~IION FOR SEQ ID NO:4: .;
. . (i) SEQUENCE CHARACTERI~llCS:
- (A) LENGTH: 420 kase paLrs
.i (B) IYPE: nucleic acid
:< (C) STRANDEDNESS: single : -
-~ (D) I~POLOGY: linear ; .
'~. :
,
, ,
:
wo 92/11378 pcr/D~s1/oo3s6
'13~ 22
(ii) ~OL~:CUIE TYPE: cDNA
( ix~ F~UEæ:
(A) N~IE/KEY: cr~;
(B) L~TI~: 76. . 396
( lX) FE~:
(A) NAME/KEY: sig peptide
(B) ~CATION: 76. . 237
. (ix) F~E:
(A) NAME/KE~Y: mat_peptide
(B) ~lION: 238 . . 396
(xi) S~CE DESCRIPrION: SEQ ID NO:4:
:~; G~ATIC~ AAG~A~T C~A~C~A GAITACA~AC TATCA~ITC AT~CACAA~A 60
. ~ Met Lys Val ~e L~3u ~u Leu Ser Leu Ile Gly l~e
: --54 --50 --45
. ~
::` TGC IGG GCC CAA CCA TCG CTA TIG GAG TCA CTT AOG CTC Gl'l' GAr GTT 159
Cys Trp Ala Gln Pro Ser Leu Leu Glu Ser Leu Thr Leu Val Asp Val
. -40 -35 -30
GAC GCA CIG TCG GAr ~IT G~r GrA CTT GqT GAG TCT GAA AOG CIr GrG 207
:~ Asp Ala Leu Ser Asp Ile Asp Val Ieu Val Glu Ser Glu l'hr Leu Val
--25 --20 --15
; CTT GTC GAT AAC ACC ACT TTG GCT AAG AG~ TTC GTT AAC CAA CAC TTG 255
~''A, Leu Val Asp Asn Thr Thr Leu Ala Lys Arg Phe Val Asn Gln His Leu
10 --5 1 S
.;
TGC GGT TCC CA~ TIG GTT GAA ~ TTG TAC TTG GTT TGC GG~ GAA A~A 303
:, Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Axg
~ 10 15 20
-A GGT TTC TIC TAC ACT ccr A~G GCT GCT AhG GGT ATT GIC GAA CAA TGC 351
Gly Phe Fhe Tyr Thr Pro Lys Ala Ala Lys ~ly Ile Val Glu Gln Cys
~:~' 25 30 35
:............... TGT ACC TCC ATC TGC TCC TTG TAC C~A TIG GAA AAC TAC TGC AAC 396
. Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
:. . 40 45 50
~ TAGACGCAGC CCGC~GGCrC TAGA 420
. ~ .
~ (2) INFORMATION FOR SEQ ID NO:5:
'-`. (i) SEX~ CTERISTICS:
~ (A) LENGTH: 107 am m o acids
: (B) TYPE: amino acid
D) TOPOLCGY: linear ~ :
. .
.. ~
.... .
~ , ,
,., ~
~VO 92/ll378 PCT/DK9l~00396
~ ~ n (3 7 j 1
~ ~LL~ULE TYPE: prote m
(Xl) SEÇUENCE DESCRIPTION: SEO ID NO:5:
Met Lys Val Phe L_u Lu Leu Ser Leu Ile Gly Phe Cys Trp Ala Gln
- -5~ -50 -45 -40
Pro Ser Leu Leu Glu S~r Leu Thr Leu Val Asp Val Asp Ala Leu Ser
. -35 -30 -25
Asp Ile Asp Val Leu Val Glu Ser Glu Thr Leu Val L_u Val Asp Asn
-20 -15 -10
Thr Thr Leu Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser His
-5 1 5 lO
Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Ty.r
` Thr Pro L~s Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile
Cys Ser keu Tyr Gln Leu Glu Asn Tyr Cys Asn
~`, 45 50
(2) INFORMATION FOR SEO ID NO:6:
(i) SEQUENOE C~RA~ERISIICS:
(A) LENGTH: 453 base pairs
(B~ TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLCGY: linear
, . .
(ii) MoT~0LE TY~: cDNA
. . . ~
(lx) FEATURE:
;~ (A) N~ME/KEY: CDS -
(B) L~C~IIGN: 76..420
(ix) FE~:
.. (A) N~ME/KEY: sig_peptide .
(B) LOCATION: 76..270
(lx) FEATURE:
- (A) NAME/KEY: mat_peptide
(B) LOCATION: 271.. 420 .
(xi) SEQVENCE DESCRIPTION: SEQ ID NO:6:
GAATICATTC AAGAATAGTT CAAACAAGA~ GAIIACAAAC IP~CaPIqlC A~ACACAAT~ 60
~ TAA~CGAITA A~AGA ATG AAA GTC TTC CTG CIG CTT TCC CTC ATT GGA TTC lll
.::~ Met Lys Val Phe Leu Leu Leu Ser Leu Ile Gly Phe
~ -65 -60 -55
.;
. 's
:: ~
. . .~. , .
. . !
' ' ', '' " '' ' ' ' ' '
w o 92~11378 PCT/DK91/003g6
q~ 3 3rl ~ ~ 24
GC T~G GCC CAA cc~ ATA GAC ACA AGA AAA GAA GGA CIA CAG CAT GAT l59
~ Cys Trp Ala Gln Pro Ile Asp Thr Arg Lys Glu Gly Leu Gln His A~p
: -50 -~5 -40
'~C GAT ACA GAA AIT rrTG GAG CAC AIT GGA AGC GAr GAG 'ITA ACC CCG 207
Tyr Asp Thr Glu Ile TPU Glu H~s Ile Gly Ser Asp Glu Leu Thr Pro
-35 -30 -25
AAr GAA GAG TAT GIT ATT GAA AGA ACT TI~, CAA GCC ATC GAT AAC ACC 255
Asn Glu Glu Tyr Val Ile Glu Ar~ rrhr Leu Gln Pla Ile Asp Asn Thr
. -20 -15 -lO
: ~CT TTG GCT A~. AGA TTC GTT AAC CAA CAC ~rG TGC GGT ~rcc CAC TTG 303
:: ~ Thr Leu Ala Lys Ary Phe Val Asn Gln His Leu Cys Gly Ser His Leu
-5 l 5 lO
GTT GAA GCT TTG ~r~c TrG GTT TGC GGT GA~ AGA GGT ~rr~ ITC TAC ACT 351
~:. Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly E~e ~e l~rr Thr
15 20 25
~". CCI AAG GCT GCT AAG GGT All GTC G~A C~ C T~ ACC l~:C AI~ ~C 399
Pro Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys
30 35 40
`:: TCC TI~ IrAC CAA TI~ G~ AAC TACT~ACT AGACGCAGCC O~GGCI~ 450
.: Ser Leu Tyr Gln ~eu Glu Asn
45 50
AGA 453
(2) ~FORM~lION F~R SEQ ID NO:7:
. .
` ~. (i) SEQIJ~CE ~RAcTERI~I~lcs:
(A) LENGTH: 115 am~no acids
~:. (B) I~: am~no acid
~ ~ (D) IOPO~GY: linear
;-:.............. (ii) ~IEa~LE TY~: protein
: .
(xi) S~QI~ICE Dl~SCP~PlION: SEQ ID NO:7:
~ Met Lys Val Phe Leu Leu Leu Ser Leu Ile Gly l~e Cys Trp Ala Gln
Y -65 -60 -55 -50
Pro Ile Asp Ihr Arg Lys Glu Gly Leu Gln His Asp ~r Asp Thr Glu
4 5 -4 0 --3 5
. :
Ile Leu Glu His Ile Gly Ser Asp Glu Leu l'hr Pro Asn Glu Glu l~r
- -30 -25 -20
Val Ile Glu Arg qhr Leu Gln Ala Ile Asp Asn l'hr mr Leu Ala Lys
. ~ -15 --10 --5
,
; Arg Phe Val Asn Gln His Leu Cys Gly Ser Hls Leu Val Glu Ala Leu
, . ,~ .,
. :.
:: .
:-i:~,
~: i ";;
~ "
, .
, , .~ . : , , : :
WO 92~11378 PCr/DK91/00396
2~9~t373l 25
l~r Leu Val Cys Gly Glu A~ Gly Phe Phe I~r Ihr ~ro IJYS Ala Ala
` 20 25 30
Lys Gly Ile Val Glu Gln Cys Cys Ihr Ser Ile Cys Ser Leu l~r Gln
Leu Glu Asn
. 50
.. .
(2) INFORMATqON FOR SEQ ID NO:8:
`` (i) S~\TCE C~PRACIERISlICS:
(A) ~: 459 base pa~rs
(B) IYP~: nudeic acid
: (C) S~ANDE~lESS: single
D) IOPO~GS~: linear
,
' (ii) MOLEC~E T~: c~NA , .
.
. .
(~x) ~E:
(A) N~ME/KEY: CDS
(B) L~CATION: 76..435
(lx) ~A~:
.; (A) N~ME/KEY: sig_peptide
``` (B) LDC~IION: 76... 276
:`', (ix) ~AIURE: '~.`.
: ~: (A) ~/KEY: mat_peptide
' (B) LDC~TION: 277.. 435
' ,
(xi) SEQUENCE DESCRIPTION: SEO ID NO:8:
. :, .
. GAATICAITC A~AATAGTT C~AACAAGAA GAII~CAAAC IaIYa~IrIC ATACACAATA 60
.-;; lAAAOGAlrA AAAGA ATG AAA GTC TTC CTG CTG CIT TCC CTC A~T GGA TTC lll
~, . Met Lys Val Phe L~u Leu Leu Ser L~u Ile Gly Phe
67 -65 -60
`. TGC TGG GCC C~A CCT GTC CCA ATA GAC ACA AGA AAA GAA GGA cr~ C~G 159
. Cys Trp Ala Gln Pro Val Pro Ile Asp Thr Arg Lys Glu Gly Leu Gln
55 -50 -45 -40
~ ,r .
~,:', CAT GAr TAC GAT ACA GA~ ATT TTG GAG CAC ATT GGA AGC GA~ GAG TTA 207
.1 His Asp Tyr Asp Thr Glu Ile Leu Glu His Ile Gly Ser Asp Glu Leu
, r -35 -30 -25
ACC CCG AAT GAA GAG IA~ GTT ATT GAA AGA ACT TTG CAA GCC AIC GAT 255
~ Thr Pro Asn Glu Glu Tyr Val Ile Glu Arg Thr Leu Gln Ala Ile Asp
i~ -20 -15 -lO
AAC ACC ACT TTG GCT AAG AGA Trc GIT AAC CAA CAC TT~ TGC GGT TCC 303 ~:
: Asn Thr Thr Leu Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser
~ -5 ~ 5 - .
.... , '~
.,,
`.,,-''' .; ' ~ . . ' ' ' , , : ' ' ., .
"`,'. ~' ' . , . ', ' ' '
`.""".' ' ' ' ' ' ~ ' ~ ' . . . '''' 1 . '
~ ' ' '''' ' ' "'. ' .' ' . ' '" : ' . ' , , '. ,
WO 92/11378 PCI/DK91/00396
,Q i 3 ~ 26
CAC TTG GIT G~A GCT 1~; TAC TTG Gl T TGC GGI` GAA AGA GGT TTC 11~ 351
His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe
10 15 20 25
TAC A ~ CCT AAG G ~ GCr A ~ GGr ATT GTC GAA CAA I~C T~ ACC TCC 399
Tyr Thr Pro Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser
.~ 30 35 4U
. .
~TC TGC TCC TIG ~AC C~A T~G GAA AAC IAC TGC A~C TAPCGCAEC 445
:~ Ile Cys Ser Leu ~yr Gln Leu Glu Asn Tyr Cys Asn
- 45 50
:,
CC~C~EGCIC IAGA 459
(2) INFORMAIION FOR SEO ID NO:9:
:~ (i) SEQUENOE CHARACTEFI~FlCS: :
(A) LENC~I: 120 ~mino acids
(B) TYPE: am m o acid
~ (D) TOPOLCGY: linear
: (ii) ~LECULE TYPE: protein
`. (xi) SEQUEN OE DESCRIPTION: SEO ID NO:9:
.~ Met Lys Val Phe Leu Leu Leu Ser Leu Ile Gly ~e Cy5 Trp Ala Gln
-67 -65 -60 -55
ro Val ~ Ile Asp Thr Arg Lys Glu Gly Leu Gln His Asp l~r ~sp
50 --45 --40
. ,`, .
. mr Glu Ile Leu Glu ~is Ile Gly Ser Asp Glu Leu Thr ~ro Asn Glu
.. --35 --30 --25 --20
, " ~
;lu Tyr Val Ile Glu Ar~ mr Leu Gln Ala Ile Asp Asn Thr Thr Leu
-15 -10 --5
..,~,
.. ,: Ala Lys Arg F~e Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu 1 5 10
. Ala Leu l~r Leu Val Cys Gly Glu Arg Gly l~e F~e Tyr ~r ~o Lys
' 15 20 25
Ala Ala 1ys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu
30 35 40 45
.;
Tyr Gln Leu Glu Asn l~r Cys Asn
::~ 50
; (2) ~FO~.lION F~R SE:Q ID NO:10: ;
`.~, (i) SE~EN~ CH~ ERISlqCS: . '~'~
'. (A) LENGIH: 408 base pairs
~. (B) TY~: nucleic acid
:. (C) SIR~ NESS: single
(D) ~)po~: linear
.~ (ii) MOT~CULE TY~: cDN~
., .
.:.-, . . . . . . . ` .
... .. -,: . .. : i , ,. , . : ," ~ - : .,
W o 9~11378 PC~ 91/003~6
2u "3731
27
` (A~ N~ME/KEY~ CDS
. (B) IOCATION: 76.. 384 :
' ' ~ (ix) ~IURE:
(A) N~ME/KEY: sig_peptide
(B) ~0~'1: 76..225
:. (lX) E~IU~:
(A) N~ME ~ : mat_peptide
(B) LDCAIqCN: 226..384
.,
(xi) SEQUENCE ~SCRIPqION: SEQ ID NO:lO:
G~TTC~IIC AAGAA~GIT C~AACAAGAA GATTAC~AAC TATCAATTTC ATAC~CAATA 60
IAAAO~ATrA A~AGA ATG AAA GTC TTC CT~, CTG CTT TCC CTC ATT GGA TTC lll
Met Lys Val Phe Leu Leu Leu Ser Leu Ile Gly Phe
-50 -45 -40 :
.. . ..
TGC I~G GCC CAA CCA TCG AIC TTG GAr TA~ GTT GAC TTG GGT GCG G~A 159
Cys Trp Ala Gln Pro ser Ile Leu Asp Iyr Val Asp ~eu Gly Ala Glu
-35 -30 -25 :.
i CqG ATC TCC AIr CGT GGG TA~ GAr AAC CTC AAC GAC GOG A~C GAr AAC 207
.~ Leu Ile Ser Ile Arg Gly Tyr Asp Asn Leu A~sn Asp Ala Ile Asp Asn
. -20 -15 -10
~. ACC ACT TTG GCT AAG AGA TTC GTT AAC CAA CAC TIG TGC GGT TCC CAC 255
.' Thr Thr Le~ Ala Lys Arg ~he Val Asn Gln His Leu Cys Gly Ser His :
- -5 1 5 10
TTG GTT GAA GCT TTG TAC TTG GTT TGC GGT GA~ AGA GGT TrC TTC TAC 303
;~ Leu Val Glu Ala Leu I~r Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr ~-
15 20 25
~ ACT CCT AAG GCT GCT AAG GGT AIT GTC GA~ CAA TGC TGT A ~ TCC ATC 351
::~ Thr Pro Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile
; 30 35 4Q
TGC TCC TTG TAC ChA TTG GAA AAC TAC TGC AAC TAGAOGCA~C COGCAGGCTC 404 .
:i Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
~ 45 50 :~ ,
.~. IAGA 408
(2) INFORMAIION FOR SEQ ID NO:11:
....
::: (i) SEQUEN OE CH~RA~ KlsTIcs:
.; (A) TFNGqH: 103 amino acids
(B) TYPE: amino acid
~:~, (D) TOPO~OGY: linear
LECULE TYPE: protein
, .,., ~ .
.-............................................... .
W O 92/11378 PCT/DK91tO0396
28
(xi) SEQUENCE DESCRIPTICN: SEQ ID N0~
Met Lys Val Phe Leu Leu Leu Ser Leu Ile Gly Phe Cys Trp Ala Gln
: -50 -45 -40 -35
Pr~ Ser Ile TPU Asp Tyr Val Asp Leu Gly Ala Glu Leu Ile Ser Ile
-30 -25 -20
Arg Gly Tyr Asp Asn Leu Asn A~p Ala Ile Asp Asn Thr Thr Leu Ala
-15 -10 -5
`; Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala
1 5 10
Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pr~ Lys Ala
` 15 20 25 30
` Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys 5er Leu Tyr
:~ ~5 40 45
Gln Leu Glu Asn Tyr Cys Asn :~
.
(2) INFOR~IIOM FOR 5EQ ID NO:12:
~:' .:
~,i (A) ~ : 372 base pairs
;,. (B) TYPE: nucleic acid
(C) SrRANDEr~SS: single
,' (D) ~PO~GY: linear
.,
(ix) F~æ:
. (A) NAME/KEY: CDS
'` (B) ~C~IION: 76... 348
(ix) FEATUgE:
.~, (A) NAME/XEY: sig_peptide
~ (B) L0CAIION: 76... 189 ,
:, (ix) FEATURE:
(A) NAME/KEY: mat_peptide ` `
(B) L0CAII~N: 190.. .348 ;~
~' (Xl) SEQUEN OE DESCRIPIION: SEO ID NO:12:
.
`N; G2~WrrCATTC PA~u~C~3Tr CAAACAAG~A Ci~rrACAAAC ~IY~4~rrrC AI~CACAAIA 60
: .-,
q~AACGAllA AAAGA ATG A~A GTC TIC CI~ ~G CIT TCC CTC Aql~ GGA TTC 111
Met Lys Val Phe Leu Leu leu Ser Leu Ile Gly Phe :
-38 -35 -30
C ~ GCC C~A CCP~ T~ CAC ACT ACC ATC GGC ACC GCA ACI GAC AAA 159
ys Trp Ala Gln l~ro Ser His Thr Thr Ile Gly Thr Ala ~r ~sp Lys
: --25 --20 --15
'`''a ~
. ~.;' ,
, ~ .
WO 92/ l 1378 P~/DK91/00396
21~ n ~
29
AAC ~ GAI A~C ACC ACT 1~ GC~ A~ ACA :L~ GIT A~C C~ C~C I~ 207
Asn Ile Asp Asn Ihr Ihr Leu Ala Lys Ar~ Phe Val Asn G~n His I~u
-10 5 ~ 5
T~C G~ TCC CAC IIG OET G~A GC~ qlG TAC 1~ GIT I~ GGr G~A A~ 255
Cys Gly Ser His Leu Val Glu Ala Leu l~r I,eu Val Cys Gly &lu Arg
10 15 20
GGT l~C TTC TAC ACT CCT A~; GCr GCI AhG GGr All (~C G~A CAA I~C 303
Gly ~e }~e Tyr Thr ~ro Lys Ala Ala Lys Gly Ile Val Glu GJn (~ys
25 30 35
TGT A~C TCC A~ TGC TCC TTG TAC CAA TTG GAA AAC ~AC TGC AAC 348
Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn l~r Cys Asn
40 45 50
. ~ .
: TAGAOGCAGC COGCA3GCIC TAA 372
.`. (2) INFORM~IION FOR SEQ ID NO:13:
, ~ :.
-~ (i) SEQVENCE C~RACIERISTICS:
A) LENGTH: 91 am m o acids
' (B) TYPE: amlno acid
: (D) TO~OLOGY: linear
~; (ii) M~LECULE TYPE~ prote m
;~ (xi) SEQUEN OE DESCRIPIION: SEQ ID NO:13:
Met Lys Val Phe ~eu Leu ~eu Ser Leu Ile Gly Phe Cys Trp Ala Gln
.~ -38 -35 -30 -25
:~ Pro Ser His Thr Thr Ile Gly qhr Ala Thr Asp Lys Asn Ile Asp Asn
-15 -10
,ii Thr Thr Leu Ala Lys Arg Phe Val Asin Gln His Leu Cys Gly Ser His
. -5 1 5 10 ~:
,.
~ Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Iyr ~ .
.. ~ 15 20 25
: ~ :
-~-' . Thr Pro Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile
: 30 35 40 :
.,; .
Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
.....
."~ :
: . .?
. ',';
~'
;~ ,
: .
.:-
:,''. :' ~ ' ' - ` :: ', .. `
:':: , . -
:' : . . :
, -:.: , . . ::.
:, ' ~
. , , , - ~ . .
WO 92~11378 r ~) PCr/DK91/00396
2 Q ~ 1 3 0
00
4 ~ 3
~ ~t 1~ ~ t ~ b~ U~ O~
_ _ __ _ ._ _
~ ~,td~7~
. DSM Deutsche Sammlung von Mikroorganismen
und Zellkulturen
-. ~ _ ~ _.
~ r~ 71 ~4~.7 ~ 0~ _ r~ ~ e~
~ Mascheroder Weg lB
.:~ D-33DO Braunschweig
~; ¦ Ac~t ~_ ~ ~~
7 December 1990 DSM 6278
:' _ _ ~ ~
In respect of those designations in which a European
~; patent i5 sought, a sample of the deposited microorga-
-~ nism will be made available until the publication of
~. the mention of the grant of the European patent or untll
.:. the date on which the application has been refused or
withdrawn or is deemed to be withdrawn, only by the
~` issue of such a sample to an expert nominated by the
: person requesting the sample (Rule 28(4) EPC).
; .
,.,,~ _ . .
.~ . ':''',
. ." ~'"~':"''
-. .
.;~
U. ~ ARAr11 ~un~ CA~t~ ~7o Uu~lt 11~ r~Uc-~
Tt .r~Clc~ to~ h~tr~ t~ .,ll ~ s~lt7r~Rtr~ 9 ~lltto~l ~rrltt~ l~r t (~ ~ ~I r~ d 1?~ I~Stc4Uon7 ~.
Acr~7~on ~ te r ol Dr.~907~1-'l
: '
~ :
~ E~ T~ ~ ~ 7r~ O~ DDUtal~Or~ r~ hl~ t~o >- t~lcl~ b~ 7 r~ 01~
~,, /~' fi)
. :,., .. ~. ._. .. ~q _______ . _
-; 'i tA~o~ 0~/ ~
.~ O T~ --`7 ~ rrK~ r~ r~ or~ l ~UrW~ . .
,.,~ ~
:~ ~ _______ _. _.. ___________ _ ___ .~
,~ ~ '~ ~
.S ~ I~ u~ 1~)
. J
''`~
