CA1211061A - Recombinant dna-molecules and methods for protein production - Google Patents

Abstract

Abstract of the Disclosure The invention relates to recombinant DNA molecules and to methods for producing proteins by means of said molecules. Parti-cularly, the present invention relates to recombinant DNA molecules which are synthesized in Bacillus strain bacteria and are charac-terized by DNA which codes for exoenzymes excreted by a bacterium of the Bacillus strain and which are present in tens of copies in Bacillus strain bacteria, as well as to recombinant DNA molecules which are modified from the above recombinant DNA molecules and are characterized by DNA containing the regulation an excretion signals of the ?-amylase gene of B. amyloliquefaciens, to which signals a gene of any protein can be joined. These recombinant DNA molecules can be used, for example, to improve the production of ?-amylase in Bacillus strain bacteria, and their modifications to produce any protein in Bacillus strain bacteria.

Description

I

This invention is concerned with recombinant DNA molecules, and methods for producing proteins by said molecules. The invention is particularly concerned with recombinant DNA molecules that are synthesized in Bacillus strain bacteria and are known to have DNA
which codes or exoenzymes excreted by them and that are present in tens of copies in Bacillus strain bacteria; as well as with recombinant DNA molecules modified from the above recombinant DNA
molecules that are known to have DNA which contains the regulation and excretion signals of the aimless gene of B. amyloliquefaciens, to which signals a gene of any protein can be joined. As will be described in the following, these recombinant DNA molecules can be used, for example, to intensify the aimless production in Bacillus strain bacteria, and their modifications to produce any protein in Bacillus strain bacteria.
Recent development in molecular biology has created new possibilities for protein production in bacteria by recombinant DNA techniques. In addition to the possibility of producing proteins of eukaryotic cells in bacteria by recombinant DNA techniques, the synthesis of the proteins of the bacteria themselves can be signify-gently improved by increasing the number of the copies of the desire Ed gene in the cell. The number of the gene copies in a bacterium cell can be increased by joining the gene to such a plasm id or virus DNA molecule as is found in the cell in several, usually 10 to 1~0, copies. The Increased number of the gene copies in a cell usually also leads to a corresponding increase in the protein synthesis expressed by the gene.
Even though several experiments of to is type have been carried out using E. golf and plasm id or virus DNA molecules no-placating in it as host bacterium, the use of Bacillus strain bacteria as hosts is only beginning ~Cryczan et at., Molecular General Gent. 177, 459-4~7, 1979; Keg gins et at., Pro. Neil. Aged.
Sat. USA 75, 1423-1427, 1978; Yenned et at., Become. Buffs. Rest Commune., 91, 1556-1564, 1979). None of the methods publicized 50 far is concerned with increasing the production of the exoenzyme ox a Bacillus strain bacterium in Bacillus strain bacteria in a manner which would allow the gene coding for the exoenzyme to be replicated by joining it to the plasm id that is present in the Bacillus strain bacterium in several copies (Part I of the invention), nor are any of the publicized methods concerned with producing proteins by a method in which the regulation and secretion signals of -the gene of the enzyme secreted by the Bacillus strain bacteria have been joined to the gene of the protein desired to be produced (Part II of the invention).
As an example of the lust part of the invention, by which the production of Bacillus strain bacterium exoenzymes can be intensified through in-creasing the number of the genes of the desired exoenzyme in the cell, the transfer of the Bacillus-~-amylase gene is presented.
This application is a divisional of our cop ending application serial No. 392,892~ filed December 22, 1981. The parent application no-fates to a method for the preparation of a selected protein or a part thereof by joining DNA coding for the selected protein or a part thereof to a bacterium gene, comprising cleaving the Bacillus amyloliquefaciens gene for aimless at a location after the excretion signal following the regulating part or at a location after a part essential with rest poet to its excretion, joining the cleaved gene toga plasm id present in Bacillus strain bacteria in several copies, the DNA sequence coding for the selected protein or for a part thereof essential to its biological activity being joined to the cleavage site by combinant DNA techniques, transforming Bacillus subtilis host bacteria with the recombinant DNA
molecule so obtained and cultivating the transformed host bacteria for producing the selected protein or a part thereof.
The parent application also relates to a recombinant DNA molecule, containing a plasm id which is capable of multiplying in Bacillus strain bacteria, the regulation part of the aimless gene of B. amyloliquefa-glens and a DNA sequence which is essential with respect to the excre-lion and to which is joined the DNA sequence coding for the amino acids in the selected protein or a part thereof essential with respect to the biological activity of the protein, the plasm id being pueblo.
According to one aspect of the present invention, there is pro-voided a method for improving the production of proteins in Bacillus strain bacteria, which comprises Joining the gene of the excreting pro-loin of a Bacillus strain bacterium by a recombinant DNA technique to a plasm id molecule present in Bacillus strain bacteria in several copies, pa transferring the Bacillus strain host bacterium with the obtainer no-combinant DNA molecule and cultivating the transformed bacteria for pro-during excreting protein.
Another aspect o-F the present invention provides a recombinant DNA molecule, in which the gene of the excreting enzyme of a Bacillus strain bacterium or a part thereof is joined to a plasm id molecule pro-sent in the Bacillus strain bacterium in several copies.
In a preferred embodiment, the gene of the excreting enzyme is an aimless gene containing the following base sequence or a part there-of: -AXE CATACGAAAA GACTGGCTGA AAACATTGAG CCTTTGATGA CTGATGATTT
TTCGGGGCGT GTATGCTTTT CTGACCGACT TTTGTAACTC GGAAACTACT GACTACTAAA

GGCTGAAGAA GTGGATCGAT TGTTTGAGAA AAGAAGAAGA CCATAAAAAT ACCTTGTCTG
CCGACTTCTT CACCTAGCTA ACAAACTCTT TTCTTCTTCT Gr.lTATTTTTA TGGAACAGAC
130 140 150 160 170 1~0 TCATCAGACA GGGTATTTTT TATGCTGTCC AGACTGTCCG CTGTGTAAAA ATAAGGAATA
AGTAGTCTGT CCCATAAAAA ATACGACAGG TCTGACAGGC GACACATTTT TATTCCTTAT

AGATE GTTATTATTT TACTGATATG TAAAATATAA TTTGTATAAG AAAATGAGAG
TTCCCCCCAA CAATAATAAA ATGACTATAC ATTTTATATT AAACATATTC TTTTACTCTC

GGAGAGGAAA CATGATTCAA AAACGAAAGC GGACAGTTTC GTTCAGACTT GTGCTTATGT
CCTCTCCTTT GTACTAAGTT TTTGCTTTCG CCTGTCAAAG CAAGTCTGAA CACGAATACA
310 320 330 aye 350 360 GCACGCTGTT ATTTGTCAGT TTGCCGATTA CAAAAACATC AGCCGTAAAT GGCACGCTGA
CGTGCGACAA TAAACAGTCA AACGGCTAAT GTTTTTGTAG TCGGCATTTA CCGTCCGACT
370 380 390 400 ~10 420 TGCAGTATTT TGAATGGTAT ACGCCGAACG ACGGCCAGCA TTGGAAACGA TTGCAGAATG
ACGTCATAAA ACTTACCATA TGCGGCTTGC TGCCGGTCGT AACCTTTGCr AACCTCTTAC

Al`GCGGAACA TTTATCGGAT ATCGGAATCA CTGCCGTCTG GATTCCTCCC GCATACAAAG
TACGCCTTGT AAATAGCCTA TAGCCTTAGT GACGGCAGAC CTAAGGAGGG CGTATGTTTC
~90 500 510 520 530 540 GATTGAGCCA ATCCGATAAC GGATACGGAC CTTATGATTT GTATGATTTA GGAGAATTCC
CTAACTCGGT TAGGCTATTG CCTATGCCTG GAATACTAAA CATACTAAAT CCTCTTAAGG

AGCAAAAAGG GACGGTCAGA ACGAAATACG GCACAA
TCGTTTTTCC CTGCCAGTCT TGCTTTATGC CGTGTT
Embodiments of the invention will be described with reference to the accompanying drawings, in which:
Figure 1 is a flow sheet illustrating the procedure of one embodiment of the invention in which the recombinant DNA molecule is prepared, isolated and characterized;
Figure 2 is a schematic illustration of the plasm id pKT~I10 and the general structure of the obtained recombinant DNA molecule;

isle 2b Figure 3 it n representntiol1 of tile lookout ye-quince fox part of tile ~lpl;a-amylrl~e gene base starting at tile cleavage site of the restriction enzyme kiwi; anal Figure 4 is a flow sheet illustrating tile prepare-lion of the recombinal1t lo muleculf! of tile l)ref;ellt invent lion contail1ing tile regulatiol1 end ex(:retioll ~sigllal~ ox the Bacillus train ~llpl~ argyle gene.
Figure 1 Slows the per~or7narlce of the sty part of tills invention Tile gnome ox the whole bacterium Is Isolated from tile Back I lust strain bacterium producing aimless, and cleave by a r~strictlon enzyme. Dot sequences of a desired length are Joined to the plasmld molecule cleaved by the restriction enzyme. ~ccordTng to this invention, the gnome of tile bacterium can be cleaved by the restriction enzyme Mbol and pub 10 can be used as the plasm id which can be cleaved by tile restriction enzyme Bamlll . I t must be noticed that a corresponding recomblnal1t DNA molecule can be prepared also by using other restriction enzyllles or plasmlds, and an experlel-ced scientist can choose between various restriction enzyme/plasmld comblr1atiol1s~ and sit 11 remain we they'll tube scope of ills Invention.
after J~slnlllg the DNA scqllel1ces with tube plasmld molecules, the obtained recomblnal1t DNA molecules are transferred Into the host bacterium, and from the population of host bacteria those ùacterlum eel 15 are screened that have received a gene coding for do_ aimless, owned to the plasmld. The screening Is based 011 the ash l eyed a l I I try of tile t fans formed go l I s to produce Amy l aye .

Bed l lust slJbtl I Is strain Is used as toe Taoist bacterium In tills Invention. When tile above mentlcl1ed recombinant DNA molecule has been transferred Into tile strain, the gene codlllg for aimless Is present In It In about I copies. Isles Increases the- aimless production of toe strain to about 500 fold, a Complied to normal B. subtllls strains. the 500-fold Increase of tile O(~amylase I:`

I

, production is due, on the one hand, to the regulation signal of the aimless gene of the B. amyloliquefaciens strain used as the initial strain being about ten times more effective than that of the B. subtilis d-amylase gene, and on the other hand, to the number of aimless genes growing 50 fold. In laboratory conditions a B. subtilis strain containing a recombinant DNA molecule produces 3 - 5 times more aimless than the B. amyloliquefaciens strain used in the isolation of the gene.
The recombinant DNA molecule is isolated from the B. subtilis strain, and characterized by restriction enzymes and definition of the base order. Figure 2 shows the pKTH10 of the obtained recomb-Nat DNA molecule the exclusive restriction enzyme cleavage sites in the aimless gene or its regulation signal, and the general structure of the recombinant DNA molecule. Figure 3 shows part of the aimless gene base order starting at the cleavage site of the restriction enzyme EcoRI.
The recombinant DNA molecule concerned in this invention consists of the regulation and excretion signals of the Bacillus strain aimless gene, and of plasm id molecules what are present in the Bacillus strain bacteria in several copies in such a manner as allows the gene of any protein to be joined at the end of the excretion signal of the aimless gene, which results in the desired protein being produced in the Bacillus strain bacterium.
The preparation of this recombinant DNA molecule is shown in Figure 4.
Most of the aimless structure gene is first removed by EcoRI restriction enzyme treatment from the recombinant DNA mole cute containing the ~-arnylase gene. The obtained DNA molecule is cleaved by the restriction enzyme and shortened by exonuclease III
and So nucleate to remove the remaining aimless structure gene, thereafter it is secured by a reverse transcripts enzyme that the ends of the molecule are double-~tranded. A DNA 1 inter molecule containing the EcoRI cleavage site is then joined to the cleaved and shortened molecule. The location of the DNA 1 inter in the recombinant DNA molecule is determined by defining the DNA base order at the joining site. The last nucleotides in the aimless structure gene are removed by DNA polymers I treatment, and the new DNA linker is joined at the end of the secretion signal of the aimless gene. At this restriction enzyme cleavage site of -the DNA
linker molecule it is possible to join the structure gene of any other protein, for example, the ~-lactamase of E. golf, or the NOAH sequence or part of it of any I, or interferon coding for amino acids. The protein coded by the joined gene will then be produced in the Bacillus strain bacterium by the aid of the regulation and excretion signals of the aimless gene.
In one embodiment, the DNA sequence coding for the amino acids in the selected protein or the part thereof essential with respect to the biological activity of the protein is joined to any of the follow-in nucleated sequences of the aimless gene:

5' COG TEA TOT GTC AT TUG COG ATT ALA ALA ALA TEA GCC
3' GAY AT ALA CRAG TEA ARC GGC TEA TOT TOT TOT AT COG
5' CUT TEA TOT GTC AT TUG COG ATT ALA ALA ALA TEA GCC G
3' GAY AT ALA CRAG TEA ARC GGC TEA TOT TOT TOT AT COG C
5' COG TEA TOT GTC AT TUG COG ATT ALA ALA ALA TEA GCC GUT
3' GAY AT ALA CRAG TEA ARC GGC TEA TOT TOT TOT AT COG CA
5' COG TEA TOT GTC AT TUG COG ATT ALA ALA ALA TEA GCC ETA
3' GAY AT ALA CRAG TEA ARC GGC TEA TOT TOT TOT AT COG CAT
5' COG TEA TOT GTC AT TUG COG ATT ALA ALA ALA TEA GCC ETA A
3' GAY AT ALA CRAG TEA ARC GGC TEA TOT TOT TOT AT COG CAT T
5' COG TEA TOT GTC AT TUG COG ATT ALA ALA ALA TEA GCC ETA AA
3' GAY AT ALA CRAG TEA ARC GGC TEA TOT TOT TOT AT COG CAT TO

I

kiwi DETAILED DESCRIPTION OF THE PERFORMANCE OF TIRE IT PART OF THE
INVENTION

Isolation, purification and cleavage of the gnome from Bacillus train bacteria B. amyloliqueFaciens strain was used as the bacterium strain.
The strain was grown over night in a rich nutrient solution, the cells were harvested and washed in a 0.0015 M odium citrate -0.015 M Nazi buffer The washed cells were suspended ( 2 x 1011 cells, i.e. a culture of 200 ml) into 2 ml of 20 % wove succors -50 my Trip - HI solution (pi 8.0). 20 my lysozyme, ZOO my prunes and 2 ml 1 w/v SarkosylR - 0.1 M ETA solution (pi owe) were added, and the solution was incubated for 15 hours at 37C. 6.5 ml H20 and such an amount of solid Shekel as to make the refraction index of the Lucite 1.4100, were added, and the Iysate was centric fused (Beckman To 50 rotor, 36 000 rum, 48 hours, 10C). The centrifuged Iysate was divided into fractions, and those fractions thaw were presumed to contain the bacterial gnome on the basis of their viscosity, were collected and dialyzed for 30 hours against a 10 my Trip - HI - 1 my ETA - 0.1 M Nail buffer (pi owe) at 4C).
The obtained gnome propriety was extracted three times with phenol, and the phenol was removed by ether extraction. The DNA was purified by centrifugation in linear 15 -I 30 w/v succors 0.1 M Nazi - 50 my Trip - HI - 1 my ETA, 0.1 % atrium laurel sulfite (pi owe) gradient; Beckman SUE rotor, 22 000 rum, for 16 hours at 22C, thereafter the gradient was Fraction Ed, and those fractions were collected whose DNA sequences were > 15 x lo Dalton and the DNA was precipitated by ethanol.
The gnome propriety of B. amyloliquefaciens thus isolated was incompletely cleaved by the restriction enzyme Mbol, and the cleaved DNA sequences were sorted out according to their size in the above succors gradient (Beckman SUE rotor, 22 000 rum, 16 hours at 22C). Those fractions whose DNA sequences were 1.5 -5 x I Dalton were harvested and the DNA was precipitated by ethanol.

Isolation and cleavage of the transfer vector by restriction enzyme The plasm id pueblo was used as a transfer vector. The plasm id was isolated and purified from the Bacillus subtilis strain SB202 as described earlier (Cryczan et at., J. Bacterial. 134, 318-329, 1978). The purified plasm id propriety was cleaved with the restrict lion enzyme Bohemia, which has only one cleavage site in tile plasm id molecule. The linearity of the plasm id molecule was controlled by gel electrophoresis.

Combination of the B. amyloliquefaciens gnome strands to the transfer vector The B. amyloliquefaciens gnome strands that had been cleaved by the enzyme Moo! and selected on the basis of their size, were mixed with the pub plasm id cleaved by the enzyme Bohemia in 10 my Trip Hal - 1 my ETA buffer (pi 8,0) in a DNA-concentration ratio of 1:3, with the total volume of 120 us and with the total DNA
concentration of 180 gel The solution was heated For 5 minutes at 65 C, and 13 us 66 my Trip HI - 6 6. my McCoy - 100 my dithiothreitol - 10 my AT buffer (pi 7.8) and 5 I T4-DNA ligate (20 Weiss units) were added to the chilled solution. The ligate solution was incubated for 3 h at 23C, and the ligation result was controlled by gel electrophoresis.

Transfer of the recombinant DNA molecule into the host bacterium A B. subtilis Lowe strain with the genotype Seiko, metB5, aureole, Amy , was used as the host bacterium. The strain was obtained from Bacillus Genetic Stock Center (Ohio State University, USA), and its phenotype Amy was mapped by bacteriogenetic methods as mutations in the structure gene of the enzyme coding for aimless. The strain was made competent, i.e. capable of receiving DNA in a manner described previously ~Anagnostopoulos et at., J. Bacterial. 81, 741-746, 1961). The recombinant DNA molecules prepared by ligation as described above, were mixed with the competent host bacteria, and the mixture was kept for 30 mix at 37C. The mixture was then spread on bacterium plates with kanamycin antibiotics to prevent the growth of all those bacteria that had not received a plasm id. The plates were kept for 30 hours at 37 C, during which time the host bacteria with a plasm id or a B. amylolique~aciens gnome strand joined to it, grew into small colonies.

Detection of host bacteria in which the e . amyloliquefaciens gene coding for aimless is joined to plasm id pub The bacterial colonies described above were replicated on new nutrient plates that were grown for JO hours at 37C. The obtained bacterial cultures were treated with ICKY solution using a method described earlier (J. Bacterial. 119, 416-424, 1974), which resulted in a white ring forming round those bacterial colonies that had received a recombinant DNA molecule containing a gene coding forc~-amylase. The corresponding colonies were collected from the original bacterium plates and the bacteria were subjected to several successive purification growths.

Isolation and characterization of the recombinant DNA molecule The recombinant DNA molecule was isolated and purified from the host bacterium by a method described earlier (Cryczan et at., J. Bacterial. 134, 318-329, 1978). The molecule was characterized by various restriction enzymes and the location of the gene coding for aimless was preliminary determined by following the inactivation of the gene when joining extra DNA
sequences at various sites of the recombinant DNA molecule. The base order of the gene coding for aimless was then determined by a method described earlier (Maxim, A. and Gilbert, W., Pro.
Neil. Aged. Sat. USA 74, 560-564, 1977).

Determination of the aimless activity _ The notified host bacterium B. subtilis WHO 6064 (Seiko, metB5), which has a gene coding for aimless in plasm id pueblo, was grown in a liquid nutrient medium (Lurid broth) by aerating at 37C. Samples were taken from the culture liquid at 2-hour intervals, from which the ohms activity was determined by Phadebas tablets.

I

.

DETAILED DESCRIPTION OF TIE PERFORMANCE OF THE END PART OF THE
INVENTION

Removal of EcoRI fragment from plasm id pKTHl0 The plasm id pKTHl0 was cleaved at the cleavage site EcoRI
(Fig. 2). The obtained DNA sequences (about 1 jug) were ligated together again in 66 my Trip - HI - 6.6 my McCoy - 100 rum dithiothreitol - 10 my AT buffer (pi 7.8), and 0.5 I T4-DNA
ligate (2 Weiss units) was added. The ligation solution was incubated for 3 hours at 23C, thereafter the competent B. subtilis WHO 6064 strain was transformed by it in a manner described above.
The cells were spread on bacterium plates containing kanamycin and grown over night at 37C. An -amylase-negative colony was screened from the obtained transform ants by ski method using starch plates, and a plasm id was isolated from the colony in a manner described earlier (Cryczan et at., J. Bacterial. 134, 318-329, 1978). The missing EcoRI - Kpnl Hindlil - EcoRI fragment in the obtained plasm id propriety pKTH29 was controlled by got electrophoresis.

Shortening of plasm id pKTH29 by exonuclease treatment The plasm id pKTH29 (100~ul, 500 jug/m1) was cleaved by the restriction enzyme Equal. After this treatment, 0.5,ul 1 M
dithiothreitol and 10 Al exonuclease ill (0.25 units, Bulbs) were added to the solution. The solution was incubated For 1 - 3 minutes at 37C, and the reaction was stopped in a 70C water bath.
The DNA was precipitated from the solution by ethanol and dissolved in a 0.3 M Nazi - 0.03 M atrium - acetate - 3 my ZnC12 buffer (pi 4.5). 10 ye Sl-nuclease (25 units/ml, Boehringer Minim) was added and the solution was incubated for JO minutes at 37C and for 10 mix a 4C. After the incubations, the propriety was extracted with phenol, the phenol was removed by ether extraction, and the DNA was precipitated by ethanol. The dried DNA was dissolved into 40 Al 10 my Trip - HI - l my ETA buffer (pi owe), and 10 Al 150 my Trip - lo my Clue - 40 my McCoy - 3.0 dithiothreitol buffer . r I

pi I 5 us dNTP Metro, in which to each nucleotide-tri-phos-plate lo my of the solution was mixed in equimolar ratio, and 2/ul reverse transcripts enzyme (Beard, 13 units/~ul), were added. The solution was incubated for 30 minutes at 37C and the reaction was stopped by incubation at 65C for 7 minutes. The DNA was purified by preparative agrees electrophoresis (LUG, Low celling Temperature), and the plasm id zones that had been dyed with ethidium bromide were cut off from the gel. The DNA was extracted from the agrees by phenol at 65C, the phenol extraction was repeated at 20C, and the phenol was removed by ether extraction. The DNA was precipitated by ethanol, the precipitate was washed with 70 % ethanol and dried.

Phosphorylation of EcoRI linker molecule and its combination to the plasm id 5 us pry AT (10 mCi/ml, 3000 Somali), jowl 600 my Trip -HI - 66 my McCoy - 100 my dithiothreitol buffer (pi 8.0) and 0.5 I T4-polynucleotidekinase were added to 1OJU1 EcoRI linker molecule solution (EcoRI linker, Collaborative Research, 50 gel The solution was incubated for 30 minutes at 37C, thereafter 5 I
10 my TO was added, and the incubation was continued For 30 mix at 37C. The dried pKTH29 propriety that had been treated with exonuclease, was dissolved into 5 us of Noah solution containing phosphorylated EcoRI-linker-molecule described above. 0 5 I 10 my AT, 0.5 I 1 my spermidine and JOY T4-DNA~ligase (2 Weiss units) were added to the solution. The solution was incubated for 3 hours at 23 C, thereafter it was diluted to 20~ul in I my Trip - Hal -1~0 my Nail - 10 my McCoy - buffer (pi 7.6). 15 units of EcoRI
enzyme (Bulbs) were added, and the solution was incubated for 12 h at 37C. The reaction was stopped by incubation at 65C for 10 minutes. The propriety treated with EcoRI was gel filtered through 1 ml Suffers 4B column. 2 my Trip - Hal - 0.1 my ETA pi 7.5) was used as elusion buffer in the filtering. The filtrate was harvest ted in 35 I fractions, and the fractions containing plasm id were identified by their radioactivity, collected and dried. The dried DNA was dissolved into 20 I 66 my Trip - HI - 6.6 my McCoy -I

10 my dithiothreitol buffer (pi 8.0), and 1.5 I 10 my AT Andy I T4-DNA-ligase were added. the solution was incubated for 3 hours at 23CJ thereafter the competent B. subtilis IH0 6064 strain was transformed by the plasm id propriety, and the cells were cultivated on bacterium plates containing kanamycin.
The plasmids were isolated from the transform ants by a method described earlier (Cryczan et at., J. Bacterial. 134, 318-329, 1978), and the plasmids were first characterized by Mel electrophoresis, thereafter their DNA base sequence at both ends of the EcoRi linker molecule was determined. In this way, the plasm id pith 38 was obtained from the plasm id pKTK 29. In the plasm id pith 38, the EcoRI linker molecule is located 90 nucleated pairs after the cleavage site of the excretion signal in the area of the b~-amylase structure gene. In order to join the linker molecule at the joining site of the excretion signal or in the immediate vicinity thereof, the plasm id pith 38 was cleaved with EcoRI. Three portions of jug of the cleaved plasm id were each suspended in 115 I 20 my Trip, 600 my Nazi, 12 my McCoy, 12 my Quick, 1 my ETA buffer (pi 8.1). 10 us BOYLE enzyme (Bethesda Research Laboratories, BURL, 40 U/ml) was added to each plasmide portion, and the tubes were incubated for 5, 6 and 7 minutes in a water bath of 30C. The reaction was stopped by adding 0.5 M
ETA, pi owe, so as to obtain a final concentration of 12 my. The DNA portions treated with BOYLE were combined, extracted twice with phenol and precipitated with ethanol. The ethanol precipitate was suspended in 75 I 63 my Tracy 6.3 my McCoy buffer (pit 8.0), and to the solution were added 5 ye 1 my date, I my dGTP, 1 my dCTP, and 1 my dTTP, and finally 5 I To polymers (PL-Biochemicals, 5 Us The solution was incubated for 80 minutes at 11C. The reaction was stopped by adding 0.5 ETA as above, and the solution was extracted with phenol and the DOW was precipitated wit ethanol.
The ethanol precipitate was dissolved in 250 I 10 my Trip, 1 my ETA buffer (pi I To 55 I of this solution were added 50~ul phosphorylated Hind ill linker molecule (BURL, 75 pool), 5 ye 660 my Trip, 100 my McCoy, 50 my dithiothreitol buffer (pi 7.5), and 10 ye To DNA ligate (BURL, 2 U/~ul). The mixture was incubated for 15 hours at 15 C and for 10 minutes at 65 C. The DNA was precipitated by adding isopropanol, the DNA precipitate was washed with 70 ethanol and, after drying in vacua, suspended in Lyle 10 my Trip, 50 my Nazi, 5 my McCauley, 5 my dithiothreitol buffer (pi 8.0). JOY of Hind ill restriction enzyme (BURL, 10 Us was added to the suspension, and the solution was incubated for 4 hours at 37C and for 10 minutes at 65C, the DNA was purified by electron pharisees, owe % LOT agrees gel (Marine Colludes Inc.), 30 V, 15 hours. The linear plasm id zone was cut off from the gel, the DNA
was extracted at 65C with phenol and was precipitated with ethanol.
The ethanol precipitate was dissolved in 35 ye 66 my Trip, 10 my McCauley, 5 my dithiothreitol buffer (pi 7.5~ to Welch was added JOY
10 my rat and 1.5 I To Diva ligate BRAILLE, 2 Pull The mixture was incubated for 3 hours at 22C and transformed into the competent B.
subtilis WHO 6135 strain, and the cells were cultivated on nutrient medium plates containing kanamycin. The plasmids were isolated from the transform ants according to a method described earlier, and the Vocation of the Hind ill linker molecule in the plasmids was deter-mined by means of DNA sequencing. In this way a series of plasmids was obtained in which the Hind ill linker molecule is located immediately after the excretion signal or in different positions after the cleavage site of the excretion signal in the area of the aimless structure gene.

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Jo The DNA sequence coding for the amino acids of any desired protein can be joined to the cleavage sites formed ivy these Hind III linker molecules whereby, as appears from the above examples, a bacterium of the Bacillus strain will produce and excrete said protein on its substrate.
A wide variety of proteins may be produced, in-eluding:
A. Antigenic_proteins of microbes and protozoa Capsule, outer membrane and Fimbria proteins from the following sources:
Bacteroides fragilis Fusoblcterium sup.
i~orcietella pertussis Homophiles influenza Yersinia enterocolitica Yersinla pests Branhamella caterwauls Escherichia golf Klebstella pneumonia Vlbrlo cholera Proteus marbles Pseudomonas arrogance Serratla marcescen5 Legion Ella pneumophila Nasser gunner Newsreel menlngltidls aye Salmonella typhimurium Salmonella tough Salmonella paratyphi B
Mycobacterium tuberculosis Chlamydia trachomatis ShTgella sup.
Protein toxins produced by the following bacteria:
Staphylococcus Ayers Pseudomonas aeruginosa Clostridium sup Escherlchia colt YersinTa pests Vibrio cholera Bordetella pertussis M-protein of the Streptococcus pudginess bacterium Excreted enzymes of Streptococcus mutant Surface proteins of the following organisms:
Plasm odium sup.
: Toxoplasma sup.
Leishmania sup. ) all phases of development Schistosoma 5pp~ ) Trypanosoma sup.
Membrane proteins of the following microorganisms:
Mycoplasma pneumonfae ~ycoplasma hsminls Contagious protein of Streptococcus sup.
Contagious protein of Staphylococcus Ayers By Anti en proteins of viruses g P
HA and NO proteins of myxoviruses (influenza A Hi H12 influenza influenza C) HO and F proteins of paramyxoviruses (parainfluenza 1-4~
Newcastle disease virus Measles virus Respiratory syncytial virus Parotitis virus Distemper virus) G protein of Rabies virus .

2~L~...f~6 14b El and E2'protelns of alfaviruses (Chikungunya, Western, Easter, Venezuelan equine encephalitis virus O'nyong-nyong virus, Semlikl Forest virus, Sindbis virus V1 and V3 proteins of flavin viruses (Dunk 1 - 4, Japanese encephalic t i 5 virus, Mite encephalitis viruses, Murray Valley encephalitis virus, Cozener Forest disease virus, looping ill virus, Omsk hemorrhagic fever virus) Surface proteins of Herman measles virus Surface proteins of Hog Cholera virus Surface proteins of Equine arthritis virus Go and Go proteins of Bunyan viruses rift Valley fever virus, Grumman hemorrhagic fever virus, California encepha-fills virus, Phlebotomies fever virus Go and Go proteins of arena viruses (Lass Fever virus, Lymphocytic kern meningitis virus) Proteins V1 - Us of picorna viruses (polio 1 - 3, Coxsackie A viruses 1-24, Coxsackie B viruses 1-6, ECHO
viruses 1 - 8, 11 - 34, Hematite A virus, Human rhino viruses 1 - 113) Surface proteins of rota viruses Surface proteins of herpes viruses (HSV 1, 2, Cytomegalo virus, Epstein-Barr virus, Equine abortion virus) VP1 - VP3 proteins of pap ova viruses (BY virus, Human wart virus) Proteins of parve viruses (Mink entreats virus, Bovine parve virus! Feline parve virus, Procaine parve virus) Structure proteins of Human hematite B virus Surface proteins of Eyeball and Mar burg viruses Hex one, pontoon and fiber proteins of adeno viruses (Human adeno viruses 1 - 33) C. Industrial enzymes Enzymes:
-aimless (I. subtl!is, malt, A. ours) Aminofacidacylase bacillus sup.) Amyloglucosidase (A. Niger Respace sup.) Bromelain annoyance I
14c Phlsine (Fig) (I ~ga1actosidase (A. Niger) p -glucanase (B. subtilis, Aspergillus spy Glucose-isomerase ~L.brevis, Neptunium Stewart-maces sup.) Glucoseoxidase (A. Niger) ilemicellulase (A. Niger Trichoderma Reese, Bacillus 5pp . ) Inverts (S. cerevisiae) Kettles (A. Niger Collegians (Clostridium histo!yticum) Xsylanase (A. Niger Trichoderma Reese, Bacillus sup.) Lactose (S. fragilis, S. Iactis~ i. golf, Asperglllus spot Lopez (Mound, Yeast) Naringinase (A. Niger Panereatln (Pancreas) Pa pain (Papaya) Pectins (A. Niger Pencil sup.) PenicilllnamTdase (Bacillus sup.) Penicllllnase (Basil lug 5pp.) Pepsin (Animal abdomen) Protozoa (A. ours, B. subtllis~
Pullulanase (Aerobacter aerogenes) Isoamylase (Escherlchia in~ermedia, Pseudomonas sup.) Ryan n (cay 1 f stowage f M. Moe, Endothia parasitical Ribonuclease (B. subtilis, Mound, A. Niger) Cellulose (A. Niger Trichoderma Reese) Streptokinase (Streptococcus iemolyticus) Trypsin (Pancreas) ., , " ..
I- . . . . : ,.

slur 14d The following Examples illustrate the invention:
Exam 1 e I
Production of the ~-lactamase eons E. golf from the Bacill_us_substitus strain The plasm id pith was opened by the Hind Gil enzyme, and to the cleavage site was joined a gene cotilng for -lactamase ox E.
golf from which the promoter and excretion signal areas had been removed. The hybrid plasm id obtained was transformed into the competent B. subtllis IH0 6140 strain by selecting the cells that had received the plasm id, on the bests of the kanamycln resistance, and the cells were cultivated on nutrient medium plates containing kanamycin. The transform ants were screened with respect to the yield by suspending each colony in 100 I I my nitrosephin, 0.1 M K phosphate solution (pi 7.0). Liquid cultures were made of the colonies which gave a positive result In the nltrosephln test (the color of the solution changed Into red) for determlna~lon of the activity of the -lactamase enzyme produced. The IH0 6140-B.
subtliis strain which had been transformed by the plasmld pith 50 was used as control. The strains were grown in a SUMS solution (Spizlzen minimal salts) to which had been added 0.5 % glycerol, 1 soluble starch, and 5 ~gfml kanamycln. The cultures were grown a 37C while shaking. About 5 hours after a logarithmic growth period ( Clout), the cultures were centrifuged 10.~00 9 5 minutes and the supernatant was recovered. The cells were suspend-Ed In 0.1 M potassium phosphate buffer (pi 7.0~ to their original growing volume. The -lactamase activity was determined in the cell and supernatant fractions by following spectrophotomctrically the dislntegrat ion of cephalotin. The follow no results worn obtained from the determination.

il~ilL~L~?~

P-lactamase activity(U/ml)-~
_ .
cells supernatant B. subtilis IH0 6140/pKTH 50 ~-lactamase 60 3000 B.subtilis IH0 6140/pKTH 50 ~10 <10

3 ill of,~,-lactamase disintegrates 1 Molly penicillin G in 1 minute at 37C

Example 2 Production Or luckiest interferon n the Bacillus subtilis strain The plasm id pith 53 was cleaved by the Hind ill enzyme, and to the cleavage site was joined the DNA sequence coding for the 7eukocyte interferon I from which the part coding for the excretion signal had been removed. The obtained hybrid plasm id was transformed into the competent IH0 6140 B. subtilis strain by selecting the cells that had obtained the plasm id, on the basis of the kanamycin resistance. The transform ants were screened by a colony hybridization method (Griinstein, M. and Hotness, DO Pro.
Neil. Aged. Sue (US) 72, 3961-3965, 1975) while using as probe the DNA coding for tune interferon, marked 125~ The bacterium colonies containing interferon-r,NA were grown in Lurid broth to which had been added 2 % soluble starch and 5 ,ug/ml kanamycin, while shaking at 3,C. The culture was centrifuged 4 hours after the logarithmic growth period (Clout) 10.000 9, 5 min. The supernatant was recovered, and the cells were suspended to their original growing columej~l a 0.9 Nazi solution. The interferon activity was determined in the cell and supernatant fractions.
The B.subtilis IH0 6140/pKTH 53 strain was used as control in toe determinations. The following result were obtained from the determinations:

, , :
, I

interferon t Activity (I.U./ml) cells supernatant B. subtilis IH0 6140/
pith 53-lF 30 200 000 B. subtiiis IH0 6140/
pith 53 Jo <20

Claims (10)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of improving the production of proteins in Bacillus strain bacteria, which comprises joining the gene of the excreting pro-tein of a Bacillus strain bacterium by a recombinant DNA technique to a plasmid molecule present in Bacillus strain bacteria in several copies, transferring the Bacillus strain host bacterium with the obtained recom-binant DNA molecule and cultivating the transformed bacteria for pro-ducing excreting protein.

2. A method as claimed in claim 1, wherein the gene of the ex-creting protein joined to the plasmid is an .alpha.-amylase gene.

3. A method as claimed in claim 1 or 2, wherein the gene of the excreting protein joined to the plasmid is the .alpha.-amylase gene of Bacillus amyloliquefaciens.

4. A method as claimed in claim 1 or 2, wherein the plasmid is pUBllO.

5. A method as claimed in claim 1 or 2, wherein the host bacterium is Bacillus subtilis.

6. A recombinant DNA molecule, in which the gene of the excreting enzyme of a Bacillus strain bacterium or a part thereof is joined to a plasmid molecule present in the Bacillus strain bacterium in several copies.

7. A recombinant DNA molecule as claimed in claim 6, wherein the gene for coding .alpha.-amylase isolated from a Bacillus strain bacterium or a part thereof is joined to a plasmid molecule present in the Bacillus strain bacterium.

8. A recombinant DNA molecule as claimed in claim 7, wherein the gene for coding .alpha.-amylase joined to the plasmid molecule originates from a B. amyloliquefaciens species bacterium.

9. A recombinant DNA molecule as claimed in claim 8, wherein the gene for coding .alpha.-amylase contains the following base sequence or a part thereof:

10. A recombinant DNA molecule as claimed in claim 6, 7 or 8, wherein the plasmid molecule used is PUBllO.