CA2084678A1 - Vector to produce biologically important peptides - Google Patents

Abstract

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.

Description

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.

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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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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 ... . .. . . . ............... , . ` :

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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.

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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.

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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 - , ~
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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~

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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

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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

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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 ~

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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~

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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 ~. , . , . , ' ,: . :

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WO 91tl980~ PCI`/SE91/00424 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~
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~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.
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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

.... . . . .
-: .

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WO 91/19805 2 0 8 4 6 7 ~ 2 2 PCr/SE91/00424 In~-m~on-l Appllc~on Nc: PCT/ ~i . .. ,_ _ __ .

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The National Collections of Industrial ~ Marine ~acteria Ltd.
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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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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 . _ .
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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).

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Claims (30)

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 .

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.

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.

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.

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.