VECTEUR POUR PRODUIRE DES PEPTIDES IMPORTANTS SUR LE PLAN BIOLOGIQUE

VECTOR TO PRODUCE BIOLOGICALLY IMPORTANT PEPTIDES

Abrégé anglais

2084678 9119805 PCTABS00008
In this patent application we have described the construction of
a novel secretion vector based on E.coli enterotoxin coding
sequence. We have shown categorically that pre and pro region of
toxin gene are absolutely necessary for extracellular secretion of
the stable toxin. We have also shown with specific examples that
when the nucleotide coding sequence of a heterologous peptide is
fused in frame to the end of the pro region in the st gene,
the resultant vector in an E.coli host secretes
extracellularly correctly processed heterologous peptide. This application
also includes construction of suitable vectors where this gene
fusion can be achieved. General methods to create such fusions
involving a) recombinant DNA technology and b) the use of site directed
in vitro mutagenesis, have also been described. A general
method of purification of heterologous peptides is also described in
this application. This novel vector system can be used for
hyperproduction and extracellular secretion of peptides of biological
importance.

Revendications

WO 91/19805 PCT/SE91/00424
24
The claims defining the invention are as follows:
1. A vector, capable of facilitating the secretion of
heterologous protein expressed in a host cell, said vector
including DNA encoding the E. coli st pre-pro sequence
<IMG> .
2. A vector according to claim 1 containing the BamHI-
HindIII segment of the E. coli st pre-pro sequence as
defined in claim 1.
3. The vector pARC 0101, Deposit No. NCIB 40115.
4. A construct comprising a vector according to any one
of the preceding claims fused in reading frame to DNA
encoding a desired protein.
5. A construct according to claim 4 wherein the DNA
encodes Angiotensin I.
6. A construct according to claim 4 wherein the DNA
encodes insulin A chain.
7. A construct according to claim 4 wherein the DNA
encodes insulin B chain.
8. The construct pARC 0726 containing the E. coli st
pre-pro sequence fused with the Angiotensin I coding
sequence in frame.
9. The construct pARC 0726 containing the E. coli st
pre-pro sequence fused with the insulin chain A coding
sequence in frame.

WO 91/19805 PCT/SE91/00424
10. The construct pARC 0726 containing the E. coli st
pre-pro sequence fused with the insulin chain B coding
sequence in frame.
11. A vector or construct according to any one of the
preceding claims substantially as hereinbefore described
with reference to the accompanying experimental data and
drawings.
12. An E. coli host containing a vector or construct
according to any one of the preceding claims.
13. A process for the production of a protein
heterologous to E. coli, by growing an E. coli host as
defined in claim 12 and isolating the expressed protein.
14. A process according to claim 13 wherein the protein
is secreted extracellularly.
15. A process according to claim 13 or 14 wherein
Angiotensin I is isolated.
16. A process according to claim 13 or 14 wherein insulin
chain A is isolated.
17. A process according to claim 13 or 14 wherein insulin
chain B is isolated.
18. A process according to any one of claims 13 to 17
substantially as hereinbefore described with reference to
the accompanying experimental data and drawings.

WO 91/19805 PCT/SE91/00424
26
19. A process for facilitating the secretion from the
host cell of a protein expressed in a host cell which
comprises fusing in reading frame DNA encoding the protein
to be expressed to an E. coli st pre-pro sequence as
defined in any one of claims 1 to 11.
20. A process according to claim 19 substantially as
hereinbefore described with reference to the accompanying
experimental data and drawings.
21. The construct pARC 0801 containing the complete E.
coli st gene sequence and containing an internal Eco RI
site, said Eco RI site having been created by using the
alternate codon for Ser 55 of the mature ST peptide.
22. A process for forming a construct which comprises
fusing a desired sequence at the Eco RI site of the pARC
0801 construct.
23. A process according to claim 22 substantially 5
hereinbefore described with reference to the accompanying
experimental data and drawings.
24. The use of E. coli st pre-pro sequence coding for M K
K S I L M I F L S V L S F S P F A Q D A K P V E S S K E K I
T K E S K K C N I A K K S N K S G P E S M in the
development of a vector for secretion of heterologous
proteins.
25. The use of the vector pARC 0101, deposit No. NCIB
40115, in the construction of an expression vector for
heterologous proteins.

WO 91/19805 PCT/SE91/00424
27
26. The use of the construct pARC 0726 containing the E.
coli st pre-pro sequence fused with the Angiotensin I
coding sequence in frame, in the expression of Angiotensin
I.
27. The use of a first construct pARC 0801 containing the
complete E. coli st gene sequence and containing an
internal Eco RI site, said Eco RI site having been created
by using the alternate codon for Ser 55 of the mature ST
peptide, as a starting material in the preparation of a
second construct by fusion of a heterologous or homologous
gene sequence at the Eco RI site of the pARC 0801
construct.
28. The use of the construct pARC 0726 containing the E.
coli st pre-pro sequence fused with the insulin chain A
coding sequence in frame, in the expression of insulin
chain A.
29. The use of the construct pARC 0726 containing the E.
coli st pre-pro sequence fused with the insulin chain B
coding sequence in frame, in the expression of insulin
chain B.
30. The construct pARC 0801, deposit no. NCIMB 40417.

Description

WO 91/19805 2 0 ~ 4 6 7 ~ PCl`/SE91/00424
A NOVEL VECTOR TO PRODUCE BIOLOGICALLY IMPORTANT PEPTIDES
Field ~f t~e jnvention:
In this patent application we have described the
constructi~n of a novel secretion vector based on R.coli
S enterotoxin coding sequence. We ~ave shown categorically
that pre ar.d pro region of toxin gene are absoluteIy
necessary fo. extra cellular secretion of the stable
toxin. We have also shown with specific examples that when
the nucleotide co~ing sequence of a heterologous peptide
is fused in frame to the end of the pro region in the st
gene, the resultant ~-ector in an E.coli host secretes
extracellularly correctly processed heterologous peptide.
This appl,cation also includes construction of suitable
vectors where this gene fusion can be achieved. General
methods to creat2 sucr. _usions involving a) recombinant
DNA technology and b` the use of site directed in vitro
mutagenesis, have also been described. A general method of
purification of he~erologous peptides is also described in
this appi~cation. This novel vector system can be used
for hyper?roduction an~ extracellular secretion of
peptides o~ b.ologi~al importance.
... . .. .
. - . . . . .
. .: . . : .: . ~
;,
., ~ . :. . .

W091/l~05 2 0 8 ~ 6 7 ~ 2 PCT~SE91/~
Back~round of the inve~tiOn ~nd ~rior art:
Secretory as well as many membrane proteins are initially
synthesized as nascent intracellular preproteins with a
signal peptide attached to the N-terminus. The signal
peptide enables the protein to cross the inner membrane
barr.ier. In ~his process the protein gets cleaved and
released as a mature protein which normally resides in the
peripl~smic space. Some exceptions to this mechanism are
certain membrane proteins whose signal peptides remain
uncleaved ~S.Letenhardt, et al. in Protein Engineering,
Applicatio~ in Science, Medicine and Industry edt. by M,
Inouye nd R. Sharma, 1986 Academic Press, Inc., 157-171).
A fe~. prokaryotic proteins or peptides are synthesized as
.5 larae precu.sors possessin~ signal peptide~ (pre-region)
as well as pro-regions. Both segments are cleaved to yield
mature proteins or peptides. Examples include subtilin of
~acillus subtilis (C. Nishio, et al, 1983J
Bioche~.Biophys.Res.Commun. 116, 751-758) or stable toxin
2~ of Escherichia coli (P.Dwarakanath, et al, 1989, Gene
81, 2;9-~26). Enterotoxigenic E.coli (ETEC) strains cause
diarrhoea in humans by the elaboration of extra cellular
toxins olassified as heat labile (LT) and heat stable (ST)
family of toxins (R.N.Greenberg and R.L. Guerrant (1981).
~c Pharmaccl.Ther.II, 507-537; M.D. Gill and M.Woolkalis 1985
in Microti~l toxins and diarrhoeal diseses, Ciba
Founda.ion symposium 112, Pitman, London, 57-73). ST
toxirls a-e of two types: methanol solub~e toxins (ST~) and
- methanol insoluble toxins (STII). S~I is further
classified into 3 groups depending upon the origin i.e.
STh (h~mar.), STp (porcine) and STb (bovine). The genes for
~otn t~le LT and ST are plasmid encodedO The nucleotide
seguence of the st gene is shown in Fig. 1. (PODwarakanath
et al, 1989, Gene 81, 219-226). From the n~cleotide
- 3i sequer.~e as well as from the translated amino acid
seguen~e it is concluded that t~e 72 amino acid peptide is
a precurso. of ST which is processed post translationally
to releas~ l9 amino acid peptide from the carboxy ter~inal
S5~ ;TU~ T
~ -

WO91/t9805 PCT/SE91/~
as the biologically active toxin. (P.Dwarakanath et al,
1989, Gene, 81, 219-226).
Attempts have been made by many groups to utilize the
"signal sequence" portion of naturally occuring secretory
proteins to construct recombinant vectors that will
secrete heterologous proteins. In many cases synthetic
signal sequences are also used. In all these case~ the
recombinant products are localized within the subcellular
compartments. Specific examples are: A patent by Gray et
al (US patent 4,755,465 July 5, 1988) where the
inventors have claimed that they have constructed a vector
which promotes the secretion of correctly processed human
growth hormone (hG~) in E.coli and Pseudomonas. The
"signal sequence" which also comes under the patent claim
1~ is very different from E.coli stable toxin "signal
sequence".
II. A second patent (~5 patent No. 4,704,362 dated Nov.
3, 1987) by Itakura et al. descxibes a recombinant cloning
2~ vehicle for microbial polypeptide expression where a
fusion product of B-gal and somatostatin is produced and
then processed ln vitro to get the final product.
An example where specifically an enterotoxin signal
sequence is employed, is by Gray et al. (US patent 4,680,
262,1985) where the inven~ors linked methanol insoluble
stable toxin (STII~ "signal sequence" with human growth
hormone (hGH) gene and localized the product in the
periplasmic region of the host cell. It is interesting to
note that inventors were specifically looking for an
expression vehicle which localized the expressed
recombinant pro~ein intracellularly. The st II signal
sequence bears no similarity to the st I signal sequence
and therefore it is considered a different structure.
In this patent applicati~n the inventors have taken
advantage of both the pre and the pro region of STI to
crea.e a recombinant vehicle in which the nucleotide -
.
. . , . , -
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~ ' ' .~' :. '' . ~ ' ' :
.
,: . ,

WO91/1~05 2 0 8 4 6 7 8 PCT/SE91/~
sequence coding for a peptide is fused inframe at the
terminus of the pro region- Expression of the whole gene,
resulted in the secretion of the recombinant peptide
extracellularly and it was correctly processed. A
schematic diagram of the principle is shown below:
- Signal seq. 1 1 Mature - -
Pre region 1 Pro r~ion 1 ST peptide
Vector _
Signal seq~ Mature
Pre region 1 Pro region 1 heterologous
peptide
Vector __ _
The advantage of this system is that the purification of
the recombinant product becomes much simpler as the cells
harboring such a recombinant vector can be grown in a
synthetic medium and therefore the secreted peptide
constitutes the major peptide present in the culture
supernatant. A general method of purification of such a
recombinant heterologous peptide (AngiotensinI) is
decribed here.
Summary:
Utilizing the pre and the pro region of E.coli st I gene
of the human variety a novel recombinant vector has been
constructed which can properly process and extracellularly
secrete any peptide. This process is achieved by fusing a
coding sequence to the terminus of the pro region. A
generalized method of purification of such a recombinant
peptide, is described in this application.
The present invention is summarized as follows in the
following clauses:
~;UOE~ ~ ITU~ 51~EE

WO 91/19805 ~ O 8 ~16 7 8 PCI/SE91/00424
s
l. The use of E.coli st pre-pro sequence coding for
(single letter code) M R K S I L M I F L S V L ~ F S
P F A Q D A ~C P V E S S ~ E R I T K E S K K C ~ I A R
K S N K S G P E S M in the development of a vector
S for secretion of heterologous proteins.
2. The vector~pARC 0101, deposit no. NCIB 40115.
3. An E. coli host çontaining the vector pAXC Q10
according to clause 2.
4. The use of pARC 0101 according to clause 2 ir. t.he
construction of an expression vector for heter~iogous
proteins.
5. A vector containing the E. coli st pre-pro sequence
as defined in clause 1.
, 6. A vector accordin~ to clause 5, containing the ~m
HI-Hind III sequence of the E. coli st pre-pro
sequence as defined in clause 1.
7. A E. coli host containing a vector as defined in any
of clauses S and 6.
~5
8. A process for the production of heterologous p~cte ns
in E. coli, by growing, by standard methods, a~ ~.
coli host as defined in clause 7 and isolat_r.g, by
standard methodsr the desired prctein product.
9. A process according to clause 8 wherein the ~roteir.
product is secreted extra-cellularly.
10. The construct pARC 0726 containing t~e E. cGli. ct
pre-pro sequence fused with the Angiotensin I co~ n~
sequence in frame.
11. The use of the construct pARC 072~ according to
~1 $E:5~ETI~TE S~E r
... . .. . . . ............... , . ` :
. .: . . .:
: . : - . .

WO91/1~ 2 0 8 4 6 7 ~ PCT/SE91/~
clause ll in the expression of Angiotensin I.
12. An E. coli host containing the construct pARC 0726
according to clause lO.
s
13. A process for the production of Angio~ensin I by
growing an E.coli ~ost according to claim 12 and
isolating the desired protein product.
14. A process according to clause 13 wherein the protein
product is secreted extra-cellularly.
15. The construct pARC 0801 containir.g an internal Eco RI
site, said Eco RI site having been created by using
the alternate codon for Ser 55 of the mature ST
peptide.
16. The use of a first construct pARC 0801 ~ccording to
clause 15 as a startin~ material in the preparation
of a second construct by fusion of a heterologous or
homologo~s gene sequence at the E~o RI si~e of the
pARC 0801 construct.
17. A vector, capable of facilitating the secretion of
heterologous protein expressed in a host cell, said
vector including DNA encoding the E. coli st pre-pro
sequence M K K s I L M I F L S V L S F S P F A Q D A K
P ~ E S S K E R I T K E s K K C N I A K K S N K S G P E
S M.
l8. A construct comprising a vector according to clauses
5j 6 or 17, fused in reading frame to DNA encoding a
desired protein.
5'J~SrlTUTE 8~ ET
.
. - ~

WO 91/19805 2 0 8 4 6 7 8 PCT~SE91/~
7.
19 A construct according to clause 18 wherein the DNA
encodes Angiotensin I.
20~ A construct according to clause 18 wherein the DNA
encodes insulin A chain.
21. A construct according to clause 18 wherein the DNA
encodes insulin B chain.
22. The construct pARC 0726 containing the E. coli st pre-
pro sequence fused with the insulin chain A coding sequence
n frame.
23. The construct pARC 0726 containing the E. coli st pre-
pro sequence fused with the insulin chain B coding sequencein frame.
24. An E. coli host containing a vector or construct
according to preceding clauses.
25. A process for the production of a protein heterologous
to E. col~, by growing an E. Coli host as defined in clause
24 and isolating the expressed protein. ,
26. A process according to clause 25 wherein the protein is
secreted extracellularly.
27. A process according to clauses 25 and 26 wherein ~-
Angiotensin I is isolated.
28. A process according to clauses 25 or 26 wherein insulin
chain A is isolated.
29. A process according to clauses 25 or 26 wherein insulin
chain B is isolated.
' ' ' , ' -. : . . . : -
- : .' -. , . ' '. - .
-. , .. ';

WO 91/19805 2 0 8 ~ 6 7 8 PCT/SE91/~
~3.
30. A process for facilitating the secretion from the host
cell of a protein expressed in a host cell which comprises
fusing in reading frame DNA encoding the protein to be
expressed to an E. coli st pre-pro sequence as defined in
any one of clauses 1 to ll.
31. A process for forming a construct which comprises
fusing a desired sequence at the Eco RI site of the pARC
0801 construct.
Detailed descriPtion of the invention:
Identification and cloning of the hu~ar variety of E.coli
stI gene was descri~ed in detail by P. ~h:arak~nath et al
(1989, Gene, 81, 219-226) In brief, a plasmid of ca. 100
MDa was identified in E.coli strain 86 cal which contained
the st gene. A BamHI library of the 100 MD~ ?lasmid of
E coli 86 cal was constructed in pBR 322 a..d a st gene
containing clone was identified by DNA probe
hybridization. This st gene was rurther subc~cned in
M13mpl9 as a BamHI - HindIII fragment and ~he complete
fragment was sequenced by Sanger's method.
A part of the sequence containing the open reading frame
v (ORF) of the st gene is shown in Fig. l. T~le carboxy
- , ~
,- . . , ` , . .
- , , ,~ .

WO91/19805 9 2 0 8 4 6 i 8 PCT/SE91/~M~
terminal l9 aa of the ORF coded peptide corresponds to the
sequence of the STh pept~de ~Aimoto et al l982
Eur.J.~iochem. l29, 257-263;. Four nucleotides preceding
the start codon, a sequence suggesting a putative ribosome
binding site can be identified, The coding sequence is
followed by a pair of stop cod~n (TAA) and a l5 nucleotide
stretch with dyad symmetry presumably indicating a
transcription termination signal. The ORF codes for a 72
amino acid peptide in which the l9 amino acid carboxy
terminal is the biologically active peptide. The l9 or 20
amino acid stretch at the N-terminus constitutes the
signal peptide, having tw~ basic residues [Lys2, Lys3]
following the initiator methionine, a hydrophobic stretch
of amino acids and a consens~s se~uence for signal
cleavage junction. The exact cleavage junction of the
signal peptide from the pro-ST region is not known. The
pro ST region spans uptc M~t 53 where it is finally
cleaved to yield a b olosically active peptide secreted
extracellul~rly with As~1;4 as ~}le N-terminal of the -
mature peptide.
Estimation of the STh concentration in culture
supernatants was obtained using the competitive ELISA '! ~ '
technique. This ELISA was routinely used throughout this
investigation to estimate t.he level of STh in culture
supernatant. Hyperexpressicn ~f the st gene was achieved
by subcloning the gene f-agment in T7 promoter containing
vector. In such a hyperexpression system the induction of
hyperexpression was achieved by the addition of the
inducer (isopropylthiogala~-toside) IPTG. Purificatïcn
scheme for the ST pepti~e ~ild type/mutant) wa~ according
to the method described by P.Dwarakanath et al. ( lsas;
Gene 8l 2l9-226). Amino acid se3uence analysis of the
peptide was done by an Applied ~iosystem Amino~Acid
Sequence Analyser ~odel 4~7A,. The in vitro site directed
mutagenesis method was usea to generate mutants in the pro
region as well as in the matu.e part of the toxin peptide
coding sequence followin~ s~ dard methods (Das et al
-~ ~ E _;, h " ' ~ ` E~
, ......
.. . . : :. : `

WO91~1~05 2 0 8 ~ 6 7 ~ ~ o PCT/SE91/~
1989, Proc~tl Acad.Sci. U.S.A. 86, 496-499). Similarly,
by in vitro site directed mutagenesis method the complete
mature ST p~ptide coding region was replaced by
Angiotensin ' (Ang I) coding sequence and secretion of the
5 Ang I was monitored by radioimmuno assay (RIA). The
results are described under Experimental Data Section.
ExPerimental Data
I. Construction of secretion vectors
STh was detected in the culture supernatant of an
ETEC E.c~li isolate 86 cal which was shown to harbor
a plasmid of lOC MDa. A BamHI library of the lO0
MDa pia-~mid of E.coli 86 cal was constructed in pBR
322 and the recombinant clones were chec~ed for st
gene. ~)ne of the recombinant clones w s identified as
carryi~g ~he st gene in a l.9 Kb ~3amHI fragment ~pARC
074~. A l. ~ BamHI - HindIII fragment obtained from
pARC ~74 was cloned in pBR 322 to obtain plasmid
pARC 01~1. The plasmid pARC OlOl is the starting
materia. for all further experiments. This plasmid is
deposited in National Collection of Industrial
Bacteria, Aberdeen, scotland (strain NCIB 4Gl15), under
the B~4~st Treaty, the deposit date beingFehn~y l;, 1989.
Cultu-e s~ernatants of E.coli H~ lOl harboring pARC
OlOl el~cit a positive biological response in
suckling mice for ST.
II. Construction of Ml3mPl9ss DNA ~haqe containing st
insert. :
A l.l Kb ~'iI - HindIII fragment containing st gene
w~s isolat~c from p~RCOlOl. This was subcloned in a
Ml3mpl9R~ ested with BamHI and HindIII. The
recombir,ar~ epli~atiYe form (RF) was transformed in
JMlOl an~ ~'ated in presence of X-Gal and IPTG. The
transfo mar.t white plaques were screened for s~ se~e
~;U~--T~ I LiTE ~EET
, . . . .
-
: : :

WO 91tl9805 11 2 0 8 ~ 6 7 8 PCT~SE91/MW~
and one such phage clone was identified as ~192 (~ig. ~"'
2) which when propagated ln JM131 releases stable
toxin in the culture supernatant. 0192 is the
s~artinq material for all the in vitro mutagen~sis
experiments described.
III. Construction of the hYPerexDression vector fo,
hy~erexPression of the wild tYPe or muta~lt ST~
0192 or its mutants were propagated in JM1~1 and the
RF was isolated following an overnight gro~th of the
- bacteria following a standard protocol of plas~
purification [T. Maniatis et al. 1982 Moiecul~r
1~ Cloning. A Laboratory Manual. Cold Spring Y.arbo1lr
Laboratory, Code S~ring Harbour, NY]. A B~ HindIII
fragment of 1.1 Kb was isolated and subc-oned in a T7
promoter containing vector as a BamHI-Hindl'I
fragment such that the promoter was oriente~ in the
same direction as that of the transcriptios c~ the st
gene. The hyperexpression plasmid containing the wild
type st gene was designated as pA~C 0601 (Fig 3).
TV. Exam~les_to demonstrate the essentiality o~ th~
region for extra cellular secretion.
To demonstrate the essentiality of the pro re~3ion for
ex~racellular secretion of stable toxin, t~_ m~-~ants
of the st gene were made where the mutation was
located in the pro region. In the first ex~mp.e a
mutation was made ~t the processing site whe.e the
Met53 was altered to Ile 53 using ~192 gr~wr. ,n
CJ236 (dut- ung~] strain as the template. Th2
mutagenic primer used for creatin~ this al~era~ion
3i had the sequence 5' C C T G A A A G C A ~ ~ A A T A G
T A G C 3' (ATG-->ATT). This primer (GD21) w~s
annealed to the templa~e and extende~ in ~.e Frcsence
. ' ~'

wo gl/t980S 1 2 2 0 8 4 6 7 ~ Pcr/sEgl~00424
of Sequenase and the four dNTPs- The extended chain
was llgated by T4 DNA liga~e and thls ln vitro
synthesized double stranded DNA was used to transform
JM101. The transformant pl~ue~ were screened by DNA
sequencing and the mutant clones were identified. One
such mutant clone (0GD21) was p'aque purified and
subcloned into the hyperexpression vector by the
method as outlined in experimen.al data Section III.
The resulting plasmid pf~RC 07Cl w~s used to transform
E.coli HB101. Production of extra cellular ST
(M53->I53) in an overnight culture was compared with
that of pARC 0601 grown in E.col, HB101. The result
is shown below:
_____________________________ __________________
Plasmid in H~101 ST produc~ion in uglml
_____________________________~____________~_____
pARC0601 7
pARC0701
________________________________________________
This experiment showed that a conservative change
(MetS3 ->Ile53) in the pro region reduced the level
of secretion of stable toxin by more than 85%.
The attenuation of STh secreticn was more evident
when a deletion mutant of t was constructed where
the deletion spanned from Ser48 to SerS2 in the pro
region. Experimental protocol was the same as that
described in the previous se^t~on except for the
mutagenic primer used which had the sequence 5' G C A
A A A A A A A G T A A T A A .~ A T G A A ~ A G T A G C
A A T T A C 3' (GD-li). Hype-expression plasmid
containing this mutant 5t tDel. Ser 48- Ser52) was
designa-ted pARC 0702. When t~ lasmid was
propagated in B 101, the resu~tant clone did not
produce any detectable extrace'lular STh. These
ex~mples ~MetS3 ->Ile53 and rJel. Ser48 -> Ser52)
therefore clearly showed that tke presence of intact
pro region was necessary for ex.ra cellular STh
Sl '~S~ 5HEET
.. ,, ~ . .
.
.
-;
.. . . . ..

wos1J1s8os 2 08~ 6 78
secretion.
V. Examples demon _ atin~ non-seecificity of the N-
terminus residue or the mature peDtide for correct
processin~ and extra cellular secretion of the
peptide .
To find the effect of the N-terminal residue of the
ST peptide on t1~e processing and extracellular
secretion of the ~eptide two mutants of the st were
made. In the first example, AsnS4 of the STh was
changed to C.ln54 exactly following the method as
described sec~ion III of Experimental Data. The ~ -
mutagenic primer had the sequence of 5' G A A A G C A
T G C A G A G T A G ~ A A T 3'(ATT-->CAG). The hyper-
expression plas~id containing the mutant st Asn54 -~
Gln54 was desisnate~ pARC 0732. This plasmid when
propagated in ~E.col~ KBlOl or in the hyperexpression
strain BL21-DE3 produced equivalent amount of
extracellular tox,r. compared to that produced by
strains HBlOl or BL~l-DE3 harboring pARC 0601. In
order to determine the processing site of the
extracellularly ~ecreted mutant ST (Asn54-> Gln54),
the mutant gene was overexpressed and the peptide was
purified essentiai.y following the method described
by P. Dwarakanath et al (1989, Gene 8l, 219-226). The
HPLC purification ~rofile is shown in Fig.4. The N-
terminal sequence OI this peptide revealed a sequence
Gln - Ser - Ser - Asn - Tyr.
Another similar experimont was conducted in which
AsnS4 of STh was ~ltered to His54. The resulting
plasmid con~ainir.g the mutant st was designated
pARC 0716. Fol' OW_II, overexpression of the st
Asn54->Ile54 anc purification of peptide, the N-
termina~ sequense of the peptide was determined. The
sequence data cho~ed presence of two peptides wi~h N-
terminal sequenc-s (1) His-Ser-Ser-Asn-Tyr ...and (2)
S~ ` 9 ~
, ' ' . ' ~ ~

WO 91/19805 1l 4 2 0 ~ 4 6 7 3 PCT/SE91/~
Se--Ser-Asn-Tyr.... A HPLC purificatlon profile of
~his p~tide is shown in Flg. 5.
These examples clearly demonstrate that the N-
termifius of the STh peptide (AsnS4) is not crucial
for ~st translational processing and secretion of
the ~ptide.
VI. Exa~Dle to demonstrate qeneralit~ of the stable toxin
based secretion vector
To test the generality of the st based secretion
vector, the coding region corresponding to mature ST
p~Ftid~ (ie. AsnS4 to Tyr72) was replaced in the st
aene by Angiotensin I coding sequence. Angiotensin ,
s a ~ecamer peptide which is converted from a larger
prec~rsor Angiotensinogen by Renin protease.
Anqiotensin I is further modif~ed to yield
Angiotensin II by 'Angiotensin converting enzyme'
which ~eletes the last two carboxyl terminal residues
~is-Leu~ of Angiotensin I, Angio~ensin II is known
to he a potential vaso constrictor. The substitution
of ~ngI coding sequence in place of ST coding
se~uence in the st gene was accomplished by Run~el's
met~ol (T.A.gunkel, 1985 Proc.Natl. Acad.Sci. USA 82,
488 492) with some modifications. A mutagenic primer
(GD9! was syn~hesized having the sequence 5' G T G G
T C C T G A A A G C A T G t; A C C G G G T G T A C A T
A C ~ C C C C T T C C A C C T C T T A A T A A T A T A
A A G ~ G 3'. This-62 mer primer was annealed to ss
Q~2 ~NA template grown in CJ 236. The temperature
for ar.nealing reaction was 55 C. The amount of
- te~piate used was 3ug per reaction while the pr~mer
used ~as lOng. Following annealing, the extension
rea~ri n was carried out at 37C for 4 ~rs in the
p~ese~ce of 8 units of Sequenase and four dNTPs
(fina~ ~onc. of each dNTP was lmM). The extension mix
also r.ontained 4 units of T4 DNA ligase for the
SU~ E~
.
- ,. ,;
, '~
- ~ .,: . . .

W091/19~05 1 5 2 0 8 4 6 7 8 pcT/sEsl/~
ligation of the extended primer to take place. This
complex was used to transform E.coli JM101 and the
putative clones were identified by DNA sequencing.
The Ang I codinq seguence linked st was subcloned
into the hyperexpression plasmid and the plasmid was
designated as pAR~ 0726. This plasmid when propagated
into HB101 or hyperexpression strain BL21-DE3, the
plasmid bearing strains produced AngI peptide extra
cellularly as detected by RIA. Following
hyperexpression of pARC 0726 in BL21-DE3 in M9 medium
the peptide was purified for N-terminal sequencin~.
The purification scheme is described ~elow. We believe
this purification scheme can be applied gener~lly for
other peptides.
' '
BL21-DE3 cells harboring pARC 0726 were grown in M9
medium ~250 ml x 4). When the ~ulture rea-hed an A600nm
= O.86, the cells were induced by addir,a IYTG (final
conc. 0.5 mm). After 2.5 hrs following induction, cells
~0 were harvested and 470 ml of culture cupernatant was
mixed with 30 g of Amberlite XAD-4 and al;cwed to
stand at room temperature overnig~t. Afte~ washing the
resin thoroughly with water, the bound pep.ide was
eluted with 99% Ethanol/1% acetic acid followed by 80~
Ethanol/1% acetic acid. The eluate was conce~trated by
flash evaporation and the concentrate wa~ loaded on SP
Sephadex C-25 column (10 ml) previously e~uilibrated
with 20 mM phosphate buffer, pH 6. 4. Prior to elution,
the column was rinsed with water. Elution wa~ carried
out with 50 mM triethylamine. The pH of .he e'uate was
brought to 6.0 with acetic acid. The elua~ was
lyophilised and t~e dry ~owder was recon~tituted in 1
ml water. The preparation was subject to HP'~ on ~P-8
column using an acetonitrile gradient ISolvent
~'A:0.1%TFA,-Solvent B:0.1% TFAl95% Acet~nitrile: Flow
/ rate 1 mllmin. Gradient was 10-50%B in 40 ~in~). The
Ang I peak was dete~ted at 29% B (Fig.6~. T~.= purified
peptide was sequenced and the seguence ~as ccnfirmed
~. , . , . , ' ,: . :
., ' ' ' "

WO 91tl980~ PCI`/SE91/00424
2084678 V
with the nat~ve sequence.
Thls example clearly demonstrates that a completely
heterologous peptlde codlng sequence can be linked
with st pro region and the correctly processed pept~de
can be detected extracellularly following propagation
of such construct in a suitable ~.coli host.
VII. Construction of a qeneral secretion vector with
suitable cloning site.
Introduction of a suitable cloning site in the mature
peptide coding region can ~e accomplished as follows:
To introduce a restriction site within or a~ very close -
proximity to the mature peptide coding region, the DNA
stretch 5' A T G A A T A G T 3' representing the amino
acid sequence from Met53 to Ser55 is chosen. By
altering the nucleotide se~uence of Ser55 codon (AGT)
to the alternate codon TCT, an EcoRI site could be
ge~erated without altering the amino acid residue. A
schematic diagram is shown below:
Met53 Asn54 Ser55
A T G A A T A G T
A T G A A T T C T
EcoRI site
The single stranded (ss 3 0192 DNA template is
mutagenized by the method described earlier. The
mutagenic primer (GD13) had a nucleotide sequence, 5'A
G C A T G A A T T C T A G C ~. A T T A C 3 ' . A genera
hyperexpression vector syst^m -an be Constructed
utilizing this EcoRI site as the suitable site for
insertion of heterologous ~rotein and peptide coding
sequence. The hyperexpression r,lasmid pET7 is digested
with EcoRI and BamHI and the large fragment isolated.
0192 (GD13) RF is ~solated all~ digested with ~amHI and
SIL~E3S~ t~
,
' '' . . ',. ~ , '. ' . '':'~ .. '
,
' ' . ' '

WO9l/l9805 17 2 0 8 ~ 6 :7 8 PCT/SE9t/~
~QBI. Following CIP (calf intestinal phos~hatase)
treatment of the dlgestion product, a 620 bp fragment
is isolated. This fragment is ligated with .he large
fragment of pET7. The resultant recombinant plasmid
is named pARC 0801.
The plasmid p M C 0801 is deposited under the Budapest
Treaty in the National Collection of Industrial and
Marine Bacteria, Aberdeen, Scotland under no. NCIMB
40417. The date of deposit is April 29, 1991.
~Fig. 7) This plasmid can be used as a genei-al
secretion vector. Any heterologous peptide or
protein coding sequen~e can be inserted at the EcoRI
site (insertion site), for example, peptides such as
Angiotensin I, bovine fibroblast growth factG-,
(bFGF), insulin, and others.
Expression of this plasmid in a E. 5Qll ;nost, results
in the secretion of the peptide or pro~eir. in ~ the
medium which can be purified. It should be noted
that peptide produced from this recombinant p'asmid
will have one additional amino acid residus a t its
amino terminal (Ser).
c ~,~
The possibility of producing Angiotensin ~ and,
especially, insulin, represent an importan~ a~pect of
the present invention. Purified insulin A chain and
. insulln B chaln can be used to produce insulin.
.
.
SUBS I ~UTE SHEET

W09~ 5 2 0 8 4 6 7 8 1 ~ PC~/SE~lt~
VIII. Examples to demonstrate secretion of insul~n ~ and B
.
chains from the stable toxin base secretion vector.
The following exam~le demonstrates the gen~ ality of
the stable toxin based secretion vecto~. Yal~an
insulin consists of two polypeptide chaihs A (21
amino acid residues) and B (30 amino acid e~_dues)
which are linked through SH bonds. These c~.~ins can
be separated in vitro by reduction and pure f ~rms of
A and B chains can be obtained. Under appropriate
conditions purified A and B cha,ns can be reoxidised
to form immunoreactive and biologically active human
insulin. The gene sequences Oc human insulin A and B
chains were fused separately at the end of the ST
pre- pro sequence. The recombinant plasmid harboured
in actively growing E. coli hcsts, secreted into the
culture medium substances which were immunoreactlve
against human insulin antiboiy. Incubation of ~he
human insulin antibody with authentic insulin A and B
chains inhibited in a competit.ive manner in
respective cases where recombinant A and B chains
were expected to be produced, lndicating that the
expected product was producec in each case. The
recombinant plasmids were constr~cted using the
strategy similar to that Zescri_ed in Experimental
Data VI. A ~13 mp 19 based recombinant phage DNA
(0 GD24) was used as the ~emplate for the first round
of mutagenesis. 0 GD2i had 3 ST gene inserted as a
Bam~I-HindIII fragment in Mi3 mp 19 multiple cloning
site. This parti~ular ST insert had a missense
mutation at the N-terminal resid~le of the mature
toxin peptide replacing the .~sn 54 by Gly 54. The
mutagenic primer used for in-rc,ducing the first round
of mutagenesis had the nucl~ot ~e sequence
S' C C T G A A A G C A T G G G T A T C G T G G A G
C A G T G C T G T A C A T C T A T C T G C T C A
C T G T A T T A A T A A, A T A A A 3'
SU~S~E~t i E gH-E~ ~
.. `~

WOgl/l9805 t9 2 084 6 78
This mutagenesis yielded the ph~ge 0GD26 which had
the DNA sequence correspondir.g to the N-terminal 1-14
residues of insulin A chain .;se~ in frame with the
ST pre-pro region. GD26 DNA was used as the
template for the second round of mutagenesis. The
mutagenic primer had the nucleotide sequence
5' T G C T C A C T G T A T C A G C T A G A G A A C
T A C T G C A A C T A A T A A T A T A A A 3'
Following mutagenesis, GD28 was identified which
had the compiete A chain gene fused at the C terminal
section of the ST pre-pro region. In summary, with 2
rounds o' mutagenesis the complete nucleotide
sequence of ir,sulin A chain gene was fused in frame
at the 3' end o' ST pre-pro seq~ence and thereby
completely repla~ing the mature toxin gene sequence.
~owever, rest of the 3' end sequence of the ST gene
was retained int.ac~ in the final construct. The
recombinant phage GD28 was grown in JM101 and the
RF was isolated. The RF was digested with
BamHI-HindIII and t.he 1.1 Rb fragment was cloned into
pET7 as a BamX~-~indIII fragment to yield pARC 0750.
The hyperexpress on E. coli strain 8L21-DE3 was
~ransformed with pARC07S0. In a similar manner
insulin B chain w2s also synthesised and fused at the
3' end of the ST pre-pro region. The initial
ten,plate used was GD23 which-had a ST insert N54F.
The nucleot~Qe se~uence of the first mutagenesis
primer was
5' G G T C C I G A A A G C A T G T T T G T G A A T
C A G C A T C T T T G C G G A A G T C A T C T G -
G T T G A G G C T C T T T A T T A A T A A T A T
A A A 3'

WO9l/l980~ 2 0 8 ~ 6 7 8 PCT/SE9l/~
Following mutagenesls a mutant clone was identified
(0 GD32) which had the DNA sequence corresponding to
the insulin B chain 1-16 residues fused in frame with
ST pre-pro sequence. 0 GD32 ss DNA was used as the
template for the 2nd round of mutagenesis. The
nucleotide sequence of the mutagenic primer was
5' G T T G A G G C T C T T T A T C T T G T A T G T
G G T G A A C G T G G T T T C T T C T A T A C A
C C T A A G A C A T A A T A A T A T A A A G G G
~ l
Following mutagenesis, the mutant phage 0GD33 was
isolated which had the complete ~ chain gene sequence
fused at the 3' en~ of the ST pre-pro seguence. This
-used gene insert was isolated from 0GD33 as the
BamHI-HindIII fra~ment and subcloned in pET7 to yield
~A~ 0759. E. coli hyperexpression strain BL21-DE3
w.~s transformed with pA~C0759 and the transformed
clones were screened for the presence of the plasmid.
~o checX whether the clones pARCO750 and pARCO759
2~ -~er~ secreting A and B chains respectively, ELISA
~rceedures were developed where culture supernatants
cculd be directly tested. Essential features of the
ELISA procedure are deccribed below:
-~uman ir.sulin was-purchased from Novo Industries. It
was in a highly pure form which was confirmed on
r-verse phase HPLC analysis. An aliquot of this pure
,n~ulin was reduced ~uantitatively with DTT and
carboxyamidated using iodoacetamide to form stable A
& ~ chains. A direct ELISA as well as an inhibition
~I3A for A & B chain detection were developed using
S~13^`~--USE SHEET
- ` - .
. . - . .. .
. ~ . ~ ... .. . - - : :
- . . -.
. - .. .. . ,

W09l/l9805 PCT/SE9l/~
Zl 2V8~678
antibody raised against human insulin. pARC0750 and
pARC0759 in BL21-DE3 host were grown separately in M9
medium and induced with IP~G as descxibed in the
previous examples. Following induction the cultures
were centrifuged and the supernatants were checked
for the presence of A & B chains. The yield of A and
B chains as estimated by direct ELISA were
approximately 16 ug/ml and 30 ug/ml respectively.
Inhibition ELISA studies also confirmed the
quantitative estimation or the secretion level of the
A & B chains respectively.
The matter contained in each of the following claims
is to be read as part of the general description of
the present invention.
S~ S--~TlIT~ SH~ET
.... . . . .
-
: .
: .

WO 91/19805 2 0 8 4 6 7 ~ 2 2 PCr/SE91/00424
In~-m~on-l Appllc~on Nc: PCT/ ~i
. .. ,_ _ __ .
MICROOR5ANISMS
0~ Sl~_ In e~nn cUoo ~ ~h~ ~ 10 . ~_ 24 ol u~ on I
~ ID~lln~CAS10~ or o~ro~ ~
r.,~ ~ u bl~d ~n n ~IIIIo ~1~
_
_d~ln~n~
The National Collections of Industrial ~ Marine ~acteria Ltd.
.__
A~ t ~ r~r1 h~n ~l~h~ _ol cs~ ~ eo~) ~
23 St Machar Drive,
Aberdeen AB2 1RY, Scotland,
.. _ .. ~
D~ ~ Aec_~n ~
15 February 1989 NCIB 40115
... . I
. ~ODITIOI~AL I~IDICAT1011~ ue~h). Thl~ In~cnn~lon b eon~nu~ n e ~r~ ~1-1_1 0
In respect of those designations in ~hich a European patent is sought, a - .
sample of the deposited microorganism will be made available until the pub-
lication of the mention of the grant of the European patent or until the
date on ~hich the application has been refused or withdraun or is deemed to
be withdrawn, only by the is~ue of such a sample to an expert nominated by
the person requesting the sa~ple. (Rule 2C(4)EPC).
:~
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C DL l--hAT~D ~I~TL--1101~ ~lltllC~ IXUlC~nO~ ~I~T ~D~ ~ tît tb~ ~)
.
D. Jr~ fUlt~ 11110 O~ l-lDIC:~T1011~ b~11~t~b)
. . - - - . - - , . .
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W O 91/t~805 2 0 8 4 6 7 8 PcT/sEg1ton424
Int~m ~on~l Appllo~on No:PCT/ Sf ~/ I o~4~4--
MICROORGANISMS ~
O~n~l Ilh~ h conn~on ~n Ih~ ~c~oroullu~ o ~ ~ 1 7 ~n~ 8 _ 9 O~ t ~ ~ eErl~nD~ 1 5 J U L l991
. _ .
~nr~cA~o~ orD~ro~ ~ r C I
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_ ol ~_~ ln~n ~
The National Collections of Inudstrial ~ Marine Bacteria Ltd.
o~ ~ in~n ~neld~o ~ ee~ ~ eountr~
23 St Hachar Drive,
Aberdeen AB2 1RY, Scotland,
D~ ~ A~
29 April, 1991 NCIHB 40417
Il. AD91TU~I~AL U191C~T1011~ ~ (h~ ~11 not ~poue l~h~. Tbl~ Inhnn~n l~ eonUnud n ~ ~ ~ ~ O
In respect of those designations in which a European patent is sought, a
sample of the deposited microorgani~m ~ill be made available until the pub-
lication of the mention of the grant of the European patent or until the
date on which the application has been refused or withdram or is deemed to
be withdrawn, only by the issue of such a sample to an exper~ nominated by
the person requesting the sample. (Rule28(4)EPC).
.
C D~ON~T~D IITA~ rOI~ lllll~lCN H Dlcano~ ~ ~n~ nn l~o~ h~
. . . . . . . I
D. ~r~ W~ NII~C orl~DIc~no~ b ~ o~ nlmt ~
- - - -
~r Inole ~ N td ~lo~r ~rlU oo u_ to th- Int~n l ~r u ~or ~ tS~dt~ ~ oon~l n~ ol th- h~on-
" _e~lon N~r ol D~o~lt ")
. _ . _
Lg~,T~Ih b- t w ~ r_ rltl t~ Int~bon-l odlr;-~n ~h~n llbo llo ~- e~eud o~ U~u ~hlln~l O~eo)~l
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