CA1270779A - Dna sequence coding for a signal peptide of levansucrase and vectors containing the same - Google Patents

Abstract

ABSTRACT

DNA SEQUENCE CODING FOR A SIGNAL PEPTIDE OF LEVANSUCRASE
AND VECTORS CONTAINING THE SAME

The invention relates to a DNA sequence of formula and vectors containing this sequence.

These vectors can be modified by a DNA insert coding for a determined polypeptide, preferably contiguous to the above DNA sequence. The vectors so obtained are useful for transforming B. subtilis and conferring on the host bacteria the capability of producing and excreting in their culture medium said determined peptide.

Description

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DNA sequence coding for a signal peptide of levansucrase and vectors containing the same The sucrose metabolic system is an important model for studying the regulation of gene expression and secretion in Bacillus subtilis. Biochemical and genetical studies have already been made on this system which comprises at least eight different loci [Lepesant, J.A. et al (1976) In : Schlessinger D. (Ed) Micro-biology, Am. Soc. Microbiol. Washington DC, pp. 58 -69]. It includes three structural genes specificallyinduced by sucrose. One of them, sacB, codes for the exocellular levansucrase (lvs). Among the five known regulatory loci, four control the expression of sacB.
Levansucrase is a R-D-fructofuranosyl trans-ferase (E.C. 2. 4. 1. 10) which synthesizes polymers of fructose called levans. It has been obtained in a pure state [DEDONDER, R. (1966) Methods in Enzymol., 8, 500 - 505]. It has a molecular weight of 50000 daltons; its primary structure has been determined by protein sequencing [DELFOUR, A. (1981) Doctorate Thesis Paris VII University] as well as its tertiary structure by X-ray diffraction analysis [Lebrun, E. et al (1980) J. Biol. Chem., 255, 12034-12036].
The present invention is based on the dis-covery which has been made of the existence of a DNAsequence comprised in the sacB gene, which codes for a signal peptide, more generally of the existence of a precursor of levansucrase. This signal peptide has been located among the expression products of an insert contained in a modified vector which had been used for transforming an E. coli strain, whereby said insert contained a sequence including most of said sacB gene including a regulatory gene associated therewith.
The modified vector had been obtained by inserting sized partial Sau3Al digests of the DNA of the QB 2010 Bacillus subtilis strain carrying a sacR

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mutation and secreting levansucrase constitutively, in the BamHI sites of a ~EMBL3 phage by making a first screening of the modified vector by molecular hybrid-ization with a radioactively labelled probe containing the sacB gene, by making a restriction map of the first screened modified phages, whereby a modified phage was retained as the starting phage for the study which led to the invention by comparing the different restriction si-tes obtained with those deduced by a computer compi-lation from the known aminoacid sequence of the pro-tein. The finally selected phage included the entire sacB gene, except for the last twenty-six nucleotides.
An EcoRI-TaqI fragment of about 400 bp which overlaps the NH2-terminal sequence of the protein and its putative signal sequence was subcloned into the large EcoRI-ClaI fragment of pBR322. The nucleotide sequence was determined starting from pBR322 EcoRI
or HindIII sites by the method of Maxam and Gilbert [(1980) Methods in Enzymol., 65, 449 - 559]. The de-duced aminoacid sequence was compared to that obtainedby protein sequencing by A. Delfour and showed total identity downstream the Lys residue which is the mature protein N-terminal aminoacid.
Upstream this Lys residue an open reading frame of 29 aminoacids was found starting from an ATG
codon. This sequence is characterized by the presence of a hydrophilic region of 8 aminoacids with 3 Lys residues followed by a hydrophobic stretch of 21 resi-dues with an Ala as last aminoacid.
There follows a formula (I) of the DNA and aminoacid sequences in the NH2-terminal region of levansucrase. The NH2-terminal Lys of the mature levansucrase was taken as aminoacid ~1. The site of cleavage between the signal sequence and the mature levansucrase (when the latter is excreted by its natural host) is indicated by a vertical bar.

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-2 TA~AGGA GAC~TG~CG ATG AAC AIC AAA AAG m GCA AAA CAA G~A ACG AI~
fMet Asn IZe Lys Lys Phe AZa Lys GZn AZa Thr lZe TTA ACC TTT ACT ACC GC~ CTG CTG GCA GG~ GGC GQ AC~ CAA G~G m GCG
(1) Leu Thr Phe ~hr Thr AZa Leu Leu AZa GZy GZy AZa Thr GZn AZa Phe AZa A~A G~A ACG AAC C~A A~G CCA IAT AAG GAA ACA IAC GGC ATT TCC CAT ATT
lys GZu Th~ Asn GZn Lys Pro Tyr Lys GZu Thr Tyr GZy IZe Ser His IZe ACA CGC C~T GA~ ~I~ CTG C~A AIC CCC GAA CAG QA A~A A~T G~AAG
Th~ Arg ~is Asp Met Leu GZn IZe Pro GZu GZn GZn Lys Asn GZu Lys A TGA stop eodon in the same reading frame 2 codons upstream from the ATG codon (numbered -29) of the preeursor eomprising -the corresponding 29 triplets of nueleotides eoding for the eorresponding 29 amino-aeyl residues of the signal peptide is also shown here-above.
Aeeordingly the ATG codon corresponding to the Met residue located 29 aminoacids before the N-terminal Lys residue of the mature protein may be con-sidered as the start eodon. A good putative ribosome binding site was also found 9 bp upstream from the ATG.
Preliminary sequencing results indieate that the stop eodon eould eorrespond to the end of another gene sinee it is preeeded by an open reading frame of at least 185 bp. No promoter nucleotide consensus sequence was 2Q found in this sequence.

Expression of the sacB gene in E. coli minicells To eonfirm the existenee of the preeursor and '!
to determine its length, the saeB gene was expressed in an E. coli minicell producing strain. The plasmid pLS8, whieh eontains the entire sacB gene, was obtained by Gay. P. et al from a library [J.A. Hoeh (1983) J. Baeteriol., 153, 1424 - 1431]. It was introdueed by transformation into the E. eoli ARI062 minicell :~1 - ~
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producing strain. Very few recombinants were obtained and the majority of them had lost the levansucrase ac-tivity. Two recombinant clones possessing the Lvs (levansucrase ) phenotype detected directly on plate (Lepesant J.A. et al (1972) Molec. Gen. Genet. 1l8, 135 - 160 were picked and further analyzed. The presence of an active levansucrase was confirmed by enzymatic assays on the sonicated extract (the specific activity found was about 0.l enzyme unit per mg bacterial pro-tein). Furthermore, one of the clones recovered con-tained a plasmid with a DNA insertion located in the 0.95 kb HindIII fragment at the end of the sacB locus.
This sequence is about 800 bp long and contains a PstI
site; it probably corresponds IS-I. The other clone harbored a plasmid identical to the original pLS8. The proteins of this latter clone were analyzed by SDS-PAGE
after incorporation of 135s1 L-methionine. Several additional bands were detected, compared to the pattern obtained for the extract of the cells containing the vector alone pBR325 in a distinct lane. One of these bands had a MW which corresponds to that of authen-tic levansucrase (50000), and another one had a MW of about 53000. Another gel was submitted to immunoblotting and it was observed that the same two bands reacted with anti-levansucrase antibodies, suggesting that the smaller polypeptide is the mature form and the larger one a precursor form of the enzyme.
The invention is thus more particularly con-cerned with a nucleotide sequence coding for the signal peptide of formula (2).

MET ASN ILE LYS L~S PHE ALA LYS GLN ALA THR
ILE LEU THR PHE THR THR ALA LEU LEU ALA GLY GLY ALA THR
GLN ALA PHE ALA

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7~ 7<3 - 4a-The corresponding preferred nucleotide se-quence flows from formula 1 hereabove. It is however understood that any of the triplets of the correspond-ing sequence may be replaced by another one coding for S the same aminoacid, as it can be deduced from the classical genetic code.
This nucleotide sequence is of particular significance as the major function of causing the excretion of the levansucrase by its natural host can be attributed to the corresponding signal peptlde.

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The invention is further concerned with the vectors particularly plasmids, capable of transforming B.subtilis and which contain said particular nucleotide sequence or fragment, hereafter referred to as "signal fragment" and optionally, part of the DNA sequence which follows said signal sequence in the sacg gene and, accordingly, codes for the first aminoacids of the mature levansucrase ~here-after referred to as "partial levansucrase gene").
Preferably the vector of the invention is free of said partial levansucrase gene. The latter may then be replaced by another DNA sequence coding for a determined polypeptide, particularly a polypeptide whose production by B. subtilis is sought. Advantage is then taken from the capability of said signal sequence of directing the syn-thesis and excretion by the transformed B. subtili~ of said predetermined polypeptide ~hen _.
a DNA sequence coding for said predetermined protein hasbeen substituted for the DNA sequence coding for the mature levansucrase. Thus the invention opens an additional field of use of B.subtilis for the production and excretion of determined poly~eptides or peptides as diverse as are, for instance interferon, polypeptides having immunogenic properties against various pathological microorqanisms, i.e. viral hepatitis B, for instance a ~olypeptide having lmmuno~enic properties analogous to those of the HBsAg anti~en, or the so-called crystal protein of Bacillus Thurin~iensis, that is a vrotein having ~ctent insecticidal properties.
When the partial levansucrase ~ene is present, the vector of the invention then consists of an intermediate vector, then capable of being modified by an insert coding for said determined polypeptide, particularly by substitu-tion of the appropriate DNA sequence for said partial levan-sucrase gene. Preferred methods for carrying out said substitution will be disclosed hereafter.
Preferably said partial levansucrase gene contains not more than the 112 first base pairs of the gene sequence coding for the mature levansucrase gene, the 112th base ' . , :,.

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~27~79 pair being located within an EcoRI site in the natural gene sequence.
Advantageously the DNAs and vectors of this invention also possess the endogenous ribosome binding site which, in the natural sacB gene has been deemed to be located 9 base pairs upstream from the ATG first codon of the signal fragment, i.e. a AAAAAGGAG se-quence. Even more preferably the DNA or vectors of this invention further comprise the whole promoter region which, in the natural DNA including the sacB
gene, is within Sau3AI - EcoRI fragment containing about 750 base pairs, said fragment including the signal sequence and the 112 first base pairs of the levansucrase gene (and terminating at the EcoRI site already referred to hereabove).
It may be preferable to substi-tute the promoter region (sacRC) obtained from mutant of B.
subtilis which is constitutive for levansucrase, for the promoter region of sacR obtained from B. subtilis in which the synthesis of levansucrase must be induced, for instance in the presence of sucrose.
Another promoter region effective to control the transcription of a gene in B. subtilis may also be substituted for the endogenous promoter region normally associated with the levansucrase and precursor genes, more particularly upstream from the abovesaid ribosome binding site. Examples of such promoter regions are those normally associated with the endocellular sucrase coded for by the sacA gene or the promoter normally associated with the gene coding for the crystal protein in B. thuringiensis.
Preferred vectors according to the invention are those which further comprise a replicon enabling them to be replicated also in E. coli. Such vectors may accordingly be amplified in E. coli.

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Thus preferred vectors of the invention will comprise the abovesaid Sau3AI-EcoRI sequence, such vectors being further modifiable by insertions therein of an~ appropriate DNA sequence coding for a selected predetermined polypeptide. Needless to say that any part of said Sau3AI-EcoRI sequence which is of no effect on the capability of the signal sequence to be transcribed or translated can possibly be deleted.
It will however be of particular advantage to modify it in such manner that said appropriate DNA
sequence be inserted close to the terminal CGC codon of the signal sequence, for instance by the insertion of an appropriate linker including a suitable restriction site within the partial levansucrase gene. Most preferably however the insertion of said appropriate DNA sequence ought to take place contiguous to and immediately downstream from the signal sequence. This particular mode of insertion must be considered when the determined polypeptide sought shall be for subse-quent pharmaceutical use. As a matter of fact it isthen indeed most preferable that the excreted poly-peptide be free of even but a short N--terminal peptide, which would be foreign to the determined polypeptide.
It is well known that the presence of even but one additional aminoacid (or the substitution of another distinct aminoacid for the first natural N-terminal aminoacid occurring in the polypeptide sought) may damage the prospect that the polypeptide so modified be useful for therapeutical purposes.
One manner of achieving the recombination of the signal sequence and of said appropriate sequence coding for the predetermined protein under such strict contiguous conditions involves, starting from a vector comprising the abovesaid Sau3AI - EcoRI sequence, sub-jecting the plasmid to an EcoRI treatment to linearize the plasmid, then subjecting the linearized plasmid to , ... .:. . .

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- 7a a treatment with an exonucleolytic enzyme such as Bal31 under conditions controlled so as to remove the 112 base pairs of the partial levansucrase gene, then ligating the appropriate gene coding for the determined polypeptide or corresponding insert to the terminal base pair of the signal sequence by means of a ligase, such a T4 DNA ligase, either simultaneously with the preceding operation or subsequent thereto, recircular-izing the plasmid to provide a final vector containing /

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~ 7 said insert and capable of transforming B.subtilis and __ rendering the latter capable of excreting the determined polypeptide in its culture medium.
It may be noted that the correct removal of the 112 nucleotide base pairs of the partial levansucrase gene may have to be checked prior to the subsequent liga-tion procedure, i.e. by recloning the exonucleolytically-treated fragments, recovering the cloned fragments and analyzing them, such as by the Maxam and Gilbert sequencing method,thereby identifying the fragment includin~ an intact signal sequence freed of up to the ~irst nucleotide of the partial levansucrase ~ene. The identified fragment can then be used in the subsequent ligation step mentioned hereabove.
According to a s.econd alternative the starting vector can be hydrolysed with the HhaI restriction enzyme to linearize the plasmid at the level of the Ala residue in the -6 position, with respect to the terminal -1 Ala, i.e. in the corresponding GCGC site, and then ligating to the so formed extremity a synthe-tic polynucleotide of 17 base pairs to restore the final part o~ the signal sequence coding for the C-terminal Ala-Thr-Gln-Ala-Phe-Ala sequence of the signal peptide, the so restricted signal sequence then bei~g li~a-ted to the appropriate DNA insert as disclosed hereabove.
The synthetic polynucleotide sequence is either identical to or different from the corresponding natural seauence, yet with the proviso that it codes for the same peptidic sequence, for instance with a view of provoking an appropriate restricting site at the level of the codon coding for the Ala in the -1 position.

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A still further alternative for producing a vector in-cIuding an appropriate DNA sequence can be obtained by ligating the linearized vector by the 11haI enzyme as discussed in relatior. tc the second alternative (and missing the 17 last nucleotides of the signal sequence) with the apprcpriate DNA, hcwever previously mcdified wit11 a synthetic polynucleotidic sequence corres~onding to the missing part of the signal sequence upstream from the starting codon of said appropriat~ DNA and provided with the additional nucleotides in the direction opposite to that of the transcription subsequent and so selected as to enable the reconstitution of the HhaI site, by diges-tion with the endonuclease.
Needless to say that other restriction sites may lS be used for achieving similar reccmbinaticns. For ins-tance the preceding second, third or fourth alternatives may be carried out upon using the MnlI site formed in the nuclectide double-ccdcn coding for the Gly-Gly dipeptidic sequence (aminoacids at the -8 and -7 pcsi-ticnc).
It will thus be unders.tood that the different alternatives which have been indicated hereabove are but illustrative of the many embcdiments which can be contem-plated by the man skilled in the art to achieve similar results.
Further aspects of the inventicn will further appear as the description of the preferred vectors of this invention proceeds, ~articularly in relation to the drawings in which :

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t~ 3 - fig. 1 is a restriction map of an HindIII-EcoRI ragment which is used below to constr~ct plasmid pBS610 and which includes a DNA sequence of the invention; and - ~ig. 2 and fig~ 3 are diagrammatical representatives of the two plasmids constructed below.

l. Construction of plasmid p~S610.
DN~ extracted f,rom P. su'ctilis strain 168, available at the "Bacillus ~enetic Stock Center" of the Department cf Microbiology of the Ohio State Vniversity, Cclum~us, Ohio, was ~artially digested with HindIII. After polyacry-lamide gel electrophoresis the 4 kb fragments were clcned in plasmid pUC8 disclosed ~y I. Vietra and J. Messing, ~ene, 1982, 19, 259-268. Plasmid pUC8 replicates only in E. ccli. I`he transformant plasmid was recovered from the clone which was found tc synthetise levansucrase. The l~tter was detected by its action on sucrose, whic)- is hydrolyzed into glucose anc fructcse polymers (levans) under the action of levansucrase.
The plasmid recovered was digested with EcoRI and PamHI. The ~ccRI-~aml~I fragment which was isolated encomp2ssed a ~lindIII-EcoRI fragment of about 2 kb. The restricticn map of the latter fragment is shown diagrair-~.atically in fig. 1. It contained the DNA sequence of the invention. The said DNA sequence, shcwn as a thicke-ned line in fig. l, is terminated by the EcoRI site at the level of the ll2th nuclectide of the levansucrase gene. The Hihal site shown is at the level of the codon . ..

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The abovesaid BamHI-Eco~I fragment was then re-combined into a new plasmid with the BamHI-EcoRI
fragment of 4 k~ obtained from pBR322, after digestion of the latter with the BamHI and EcoRI enzymes, said last mentio~ fragment also lncluding the ~lk~n ~.en~
responsibl~ for th~ replicaticn ef pBR322 in E. coli.
A HindIII-HindIII fragment of about 2.9 kb, consisting of plasmid pC194, and including the repliccn elements which enahle said pC194 to re~licate in B. subtilis was inserted in the recombinant plasmid previously obtained,more particularlyinthe HindIII-site contained in the BamHI-EcoRI fragment including ~he DNA ~equence of the invention, to finally ~rovide plasmid pBS610, diagrammatlcally re~resented in fig.2.
Parts shcwn b~ arcC a and 'c (of about 2 kb and 20 bas~
~airs respectively originate from the last m~ntio~
BamHI-EcoRI fragment, part shcwn by arc c ~of abou 2.9 kb) ~rom pC194 and part d (of abcut 4 kb) Irom pBR322. The fragments frcm which p~S610 is form~d are also designated by the names of Ihe plasmi~s Gr r)NAs from whlch they were r~s~ectively obtaine~. pBS610 (comprisln~
abcut 8,9 kb) can be re~licated ir, both E. coli and ~ u~ s.
T~:~ plasmid pC194 can be obt~lned from plasm1ci pHV33. E. coli strin (SK 15 92) transfcrmed by pHV33 has been deposlteci at the "Coll~ctlon Na~ionale des Cul~res de ~5icr~-organismes~
(C.~;.C.~ 'aticn~l Col.cct1cr. c~ Cuitur~s of ~licro-orgar.isms) cf thc ~ateul- Irstltut~ c,f Pa.rls, Francc-, under nu~ber I-191, as mer,t1oned ln th~ pu~lished European Patent Application 83 400826 published Nov. 2, 1983.

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'7V~7~3 2. Constructicn of pBS620 A HlndIII-EcoRI fragment, containing the DNA
sequence of the invention, analcgous tc that contemplated in the preceeding example, was obtained by digestion in the presence of the EcoRI and HindIII endonucleases of the mGdified phage cbtained by the modification of the ~-E~l~L3 phage referred to in the beginning cf this disclo-sure. It comprises the sacRC promoter region of a B.
subtilis synthetizing levansucrase constitutively. A
strain of E. coli containing the so-mcdified phage has been deposited at the C.N~C.M. on July 6th, 1984, under accession Nr. I-314.
The EcoRI-~HindIII fragment of 2 kb containing ~.e D~IA sequence of this invention and the S,acRC
locus was recombined with -the EcoRI-HindIII fragment of about 4.3 kb isolated frcm pBR322 and ccntainins the replicon elements h~hich enable pBR322 tc replicate in E. coli. The plasmid obtained was digested again with HindIII and recombined with the fragment of 2.9 kb already mentioned above, from pC194, tc provide plasmid pBS620 (of about 9.2 kb) diagrammatically shown in fig.3.
The different parts cf plasmid pBS620 are identified in fig. 3 with reference to the plasmids or DN~s from which they were respectively obtained. Like paS610, pBS620 can be replicated in both _. subtilis and E. coli.

These plasmids can be further mcdified as disclcsed above tc substitute therein an appropriate DNA sequence codir.y for a determined polypeptide, preferably contiguous tc the signal sequence, fcr the partial levansucrase ger,e.
The DNA sequence ccding fcr the determined polypeptide is ,cossibly prolongated, downstream from the sequence tc be translated,by a lin~er including an appropriate restric~ior.
site, for instance an HhaI site, when recourse is had fcr . i ~
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making the modification tc the third or fcurth alt~rnatives disclcsed hereabove.
It should be understocd that the different recom-bination steps ln vitro which ha~e been referred ~ in the exa~ples, may bc carried out in any manner known per se. The different restriction enzymes which have been used are available in the trade. They c-an be used according to the manufacturers'reccm~endations.
The modified plasmids so obtained can then be used to transform ~. subtilis to cause the latter tc effectively produce and excrete the d~termined poly-peptide.
The following procedure can be used.
FGr instance B. subtilis strain QB666 (C.N.C.I~.
I-192) can ~e used. The transformatiGn may be carried out according to the known technique descri~ed by DUBNAU, ~. DAVIDOFF-ABELSON, R. SCHER, B. and CIRIGLIANO C. ~1973, J. Bacteriol. 114, 273-286). The cells ccntaining plasmids resistant to antibiotic can be selected cn SP agar plates supplemented ~ th chlo-ramphenicol (5 micrograms/ml).
The B. subtilis transformants can then be d~tected, for instance by bringing intc play antibodies previcusly prepared and active against the polypepti~e sought. The cells of the detected colcnies can then be recovered, and used f~r making cell cultures synthesi-zing and excreting the poly~eptide in the medium. The poly~e~tide may th~n be recovered from the cul~ure medium in any suitable ~anner. 1'he~-~E~3 phage has been disclosed by FRI9CHAUF A.M. et al J. Mol. Biol. (19~3) Vol. 170, pp; 827 - 842.
Another advantageous ~dification which can be brought to the final codon of the signal sequence and to the starting codon of the Levansucrase ~ene consist in the substitution - of GCC for GCG and -f GGc(coding for glycine) for A~A.
The GCG/GGC junction so formed can be cleaved as shown by the vertical line between GCC and GGCI to thereby provide corres~onding blunt extremities.
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Claims (25)

CLAIMS:

1. A DNA sequence coding for a signal peptide comprising:

2. The DNA sequence of claim 1 connected, downstream from the third nucleotide of its codon coding for the C-terminal amino acid of the signal peptide, to no more than the first 112 base pairs of an additional DNA
sequence coding for a mature levansucrase.

3. The DNA sequence of claim 1, compriming:

4. The DNA sequence of claim 3 which is free of nucleotide base pairs belonging to a mature levansucrase.

5. The DNA sequence of claim 4 which includes an endogenous ribosome binding site, AAA AAG GAG, nine base pairs upstream from its first ATG codon.

6. The DNA sequence of claim 5 which comprises an endogenous promoter region upstream from said ribosome binding site.

7. A vector, effective to transform B. subtilis, which contains the DNA sequence of claim 1.

8. The vector of claim 7 wherein the DNA sequence is free of nucleotide base pairs coding for a mature levansucrase but is connected to an additional DNA sequence; the additional DNA sequence coding for a determined polypeptide and being contiguous to and downstream from the DNA sequence.

9. The vector of claim 8 which is also replicable in E.
coli.

10. A modified B. subtilis organism which is transformed by the vector of claim 8 and which is capable of secreting the determined polypeptide.

11. The DNA sequence of claim 2, wherein a restriction site is provided between the additional DNA sequence and the DNA sequence.

12. A method for obtaining the synthesis in, and excretion from, a Bacillus subtilis of a determined protein or polypeptide by transforming the B. subtilis with a vector comprising:
1) a first DNA sequence which codes for a signal peptide having the following amino acid sequence:

2) a second DNA sequence which codes for the determined protein or polypeptide, the second DNA
sequence being contiguous to, and immediately downstream from, the first DNA sequence.

13. The method of claim 12, wherein the first DNA
seguence contains the following DNA sequence coding for the signal peptide:

14. The method of claim 13, wherein the first DNA
sequence also codes for a fragment of a mature levansucrase that is downstream from the signal peptide and is encoded by no more than the first 112 base pairs of the gene coding for the mature levansucrase.

15. The method of claim 13, wherein the vector also comprises a ribosome binding site, AAA AAG GAG, nine base pairs upstream from the first ATG codon of the first DNA
sequence.

16. The method of claim 15, wherein the vector further comprises a promoter of a sacR gene upstream from the ribosome binding site.

17. The method of claim 16, wherein the promoter is within an Sau3AI-EcoRI fragment.

18. The method of claim 13, wherein the vector also comprises a promoter selected from the group of promoters consisting of a promoter of a sacRc gene, a promoter of a sacA gene and a promoter of a gene coding for a crystal protein in B. thuringiensis.

19. The method of claim 13, wherein the vector is selected from the group consisting of pBS610 and pBS620 as shown in Figs. 2 and 3, respectively.

20. A process for obtaining the inducible synthesis in, and secretion from, a Bacillus subtilis of a determined protein or polypeptide by culturing the B. subtilis in the presence of sucrase; the B. subtilis being transformed with a vector comprising:
1) a first DNA sequence which codes for an endogenous signal peptide of levansucrase:
2) a second DNA sequence which: i) codes for the determined protein or polypeptide, ii) is contiguous to, and immediately downstream from, the first DNA sequence and iii) is in the same reading frame as the first DNA sequence; and 3) a third DNA sequence which: i) codes for an endogenous sacR promoter, ii) is upstream from the first DNA sequence, and iii) controls the transcription of the first and second DNA
sequences.

21. The process of claim 20 wherein the signal peptide has the following amino acid sequence:

22. The process of claim 20 wherein the vector further comprises: a fourth DNA sequence which codes for an endogenous ribosome binding site, AAA AAG GAG, that is nine base pairs upstream from the first DNA sequence and is downstream from said third DNA sequence.

23. The process of claim 22 wherein the first, third and fourth DNA sequences are within an Sau3AI-EcoRI fragment of said vector.

24. The process of claim 23 wherein the vector comprises pBS610 of Fig. 2.

25. The process of claim 21, wherein said first DNA
sequence contains the following DNA sequence coding for the signal peptide: