the present invention is applicable to processes for the production of any physiologically active peptides containing at least one cysteine residue . such peptides include , in addition to the α - hanp and the hpct explained in detail herein , insulin a chain and b chain , calcitonin precursor of other than human origin , somatostatin , and the like . a partner protein is any protein not containing cysteine residues . such proteins include natural proteins and a part thereof containing no cysteine residues , and modified proteins wherein cysteine residues in natural proteins or a part thereof are deleted or converted to other amino acid residues . the partner protein can contain , in addition to the above - mentioned protein , a few , for example 1 to 5 , additional amino acids . the example is polypeptides comprising 90 to 220 amino acid residues of e . coli β - galactosidase . particular embodiments of the partner protein include a polypeptide consisting of a polypeptide of e . coli β - galactosidase protein from the first amino acid at the n - terminal to 2l0th amino acid calculated from the n - terminal amino acid and two additional amino acids such as glutamic acid and phenylalanine ; a polypeptide consisting of a polypeptide of e . coli β - galactosidase from the first amino acid at the n - terminal to 97th amino acid calculated from the n - terminal amino acid and two additional amino acid residues such as glutamine and phenylalanine ; and a polypeptide consisting of a polypeptide of e . coli β - galactosidase from the first amino acid at the n - terminal to 97th amino acid calculated from the n - terminal amino acid and three additional amino acid residues such as glutamic acid , phenylalanine and leucine . these additional amino acids are introduced , for example , during the recombinant dna manipulation as an amino acid encoded by a linker dna . however , since the natural form of these polypeptides contains cysteine residues , the cysteine residues must be deleted or replaced by other amino acids . this is easily carried out by site - directed mutagenesis . any amino acid can be used for the replacement of the cysteine residues , but preferably serine is used . note , lysine is not preferable . since the above - mentioned polypeptides in natural form from e . coli β - galactosidase protein do not contain lysine residues , when a fusion protein comprising a target peptide and partner polypeptide is cleaved with lysyl endopeptidase , the partner polypeptide is not cleaved on the inside and , therefore , the target peptide can be easily isolated and purified . according to the present invention , a physiologically active peptide , i . e ., a target peptide , is expressed as a fusion protein wherein the c - terminal of the partner polypeptide is linked with the n - terminal of target peptide via a linker amino acid residue . the linker amino acid is preferably lysine , arginine , glutamic acid , or methionine . when lysine is used as a linker amino acid , the peptide bond between a target peptide and the linker lysine is proteolytically cleaved with , for example , lysyl endopeptidase to liberate the target peptide . when arginine is used as a linker amino acid , the peptide bond between a target peptide and the linker arginine is proteolytically cleaved with , for example , clostripain to liberate the target peptide . when glutamic acid is used as a linker amino acid , the peptide bond between a target peptide and the linker glutamic acid is proteolytically cleaved with , for example , protease v 8 to liberate the target peptide . on the other hand , when methionine is used as a linker amino acid , the linker methionine is degraded with cyanogen bromide to liberate the target peptide from the partner protein . a fusion protein is expressed by a gene comprising a dna coding for a partner protein such as a β - gal fragment linked in a reading frame with a dna coding for a target peptide via a synthetic double stranded oligonucleotide linker containing a codon for a linker amino acid such as lysine or glutamic acid . in this case , the oligonucleotide linker preferably contains a dna sequence coding for a part of the n - terminal of the target peptide immediately downstream of the codon for the linker amino acid such as lysine or glutamic acid lysine , and has appropriate restriction sites at both terminals thereof . the gene coding for β - galactosidase is derived from , for example , plasmid pαne2 , which contains a β - galactosidase gene under a lac promoter . a gene coding for α - hanp , a target peptide , is derived from , for example a gene coding for γ - hanp , which is a precursor of α - hanp , which gene is contained in plasmid ps224 - 3 ( european patent publication no . 0164 273 ). the gene coding for hpct is derived from , for example , plasmid pahpc38 . the promoter used to efficiently express a fusion protein gene may be any promoter of a gene for a protein produced in a large amount in e . coli or of phages . according to the present invention , a lac promoter derived from e . coli lactose gene , an lpp promoter derived from e . coli outer membrane lipoprotein gene , and a p l promoter derived from the λ phage gene are preferable . these promoters can be easily obtained from known plasmids according to conventional recombinant dna techniques . for example , according to the present invention , a p l promoter is obtained from a plasmid pp l lacz &# 39 ; 210αhanp ( fig8 ); and an lpp promoter is obtained from a plasmid pin5t4 ( fig9 ). these plasmids are described in japanese patent application no . 61 - 101100 and no . 61 - 132186 . the plasmid pp l lacz &# 39 ; 210αhanp was derived from plasmid ps20 . e . coli ( n4830 / ps20 ) containing plasmid ps20 was deposited with the fermentation research institute agency of industrial science and technology ( fri ), higashi 1 - 1 - 3 , tsukuba - shi , ibaraki , japan , on may 31 , 1984 as ferm bp - 535 , under the budapest treaty . the plasmid pin5t4 was derived from a plasmid pini - a2 . e . coli ( ja22l / pini - a2 ) containing a plasmid pini - a2 and was deposited with the fri on july 18 , 1983 as frm bp - 320 . a fusion protein of the present invention can be expressed at a high efficiency by using an expression plasmid wherein an attenuator terminator of tryptophan operon ( trp a ) is inserted immediately downstream of a structural gene for the fusion protein . for example , as shown in examples and fig4 the introduction of the trp a into plasmid pghα97 ( ser ) rop - immediately downstream of α - hanp structural gene results in the formation of a plasmid pghα97s , and the productivity of the desired fusion protein is greatly increased . gene coding for a target physiologically active peptide can be chemically synthesized . alternatively , it can be cdna prepared from mrna obtained from cells which produce the target peptide or a precursor thereof . according to the present invention , gene coding for α - hanp is obtained from plasmids pghα210rop - and pghα439rop - which contain the α - hanp gene with the lac promoter . the processes for the construction of these plasmids are described in detail in reference examples 1 to 3 . the method of cleavage between a target peptide region and a partner protein region in a fusion protein depends on the nature of a linker amino acid connecting both regions . when using lysine as a linker amino acid , lysyl endopeptidase , which splits the c - terminal of lysine residure , is used . preferably , achromobacter protease i ( api ) derived from achromobacter lyticus is used . this enzyme is commercially available from wako junyaku , japan . to obtain a target peptide , e . coli transformed with an expression plasmid containing a gene coding for a fusion protein comprising the target peptide and an appropriate partner protein is cultured in an appropriate medium , and the expression of the fusion protein is induced . cells of the e . coli are then collected and the cells are disrupted . centrifugation of the disruptant provides a precipitate containing the fusion protein . this method is advantageous in that most of contaminant proteins and peptides remain in the supernatant . the precipitate is then preferably treated with 5 to 10 m urea to solubilize the fusion protein . the use of urea is advantageous over other solubilizing agents such as sodium dodecyl sulfate ( sds ) in that urea does not inhibit the enzyme api . after solubilization of the fusion protein with urea such as 8 m urea , the treated mixture is diluted with an appropriate buffer , and api is added to the mixture to cleave the fusion protein , resulting in the liberation of the target peptide . the liberated target peptide is isolated and purified according to conventional procedures for purification of a peptide or protein . according to the present invention , since the partner protein in the fusion protein does not contain cysteine residues , a disulfide bond between the partner protein and target protein is not formed . therefore , the target peptide is isolated from the partner protein in very high yield . moreover , in an embodiment of the present process wherein the partner protein does not contain lysine residues , cleavage of the fusion protein with lysyl endopeptidase such as api does not provide simultaneous cleavage within the partner protein . therefore , the target peptide can be very easily isolated and purified . moreover , according to the present invention , since the size of the partner protein is controlled to within 90 to 220 amino acid residues , a proportion of the target protein in a fusion protein is high while maintaining the stability of the fusion protein , and therefore of the target peptide , in host cells . finally , according to the present process , since the desired fusion protein is first recovered in a undissolved fraction , i . e ., precipitate from cell disruptant , and the fusion protein is extracted from the undissolved fraction , impure proteins are effectively eliminated . moreover , when using an enzyme such as api to liberate the target peptide , this step can be carried out without further purification of the fusion protein . these procedures provide the efficient and simple production of the target peptide . the present invention will now be further illustrated by but is by no means limited to the following examples . first , processes for construction of plasmids pghα210rop - , pghα439rop - , and pp l lacz &# 39 ; 210αhanp containing dna coding for fusion protein comprising α - hanp as a target peptide and a part of native β - galactosidase protein still containing cysteine residues are described in reference examples 1 to 5 . five μg of plasmid pbr322 was digested completely with 16 units of ecori in 50 μl of hindiii buffer comprising 50 mm tris - hcl ( ph 8 . 0 ), 50 mm nacl , 10 mm mgcl 2 at 37 ° c . for 60 minutes , and the digested dna was recovered by ethanol precipitation . next , ecori ends of the dna were converted to blunt ends with 2 μl of 25 mm dntps ( datp , dgtp , dctp and dtpp ) using 4 units of t4 dna polymerase in 100 μl of ta buffer comprising 33 mm tris - acetate ( ph 7 . 9 ), 66 mm ch 3 coona , 10 mm ( ch 3 coo ) 2 mg and 0 . 5 mm dithiothreitol . next , the dna was recovered by ethanol precipitation , the dna was incubated in 20 μl of a ligation mixture comprising 20 mm tris - hcl ( ph 7 . 4 ), 10 mm mgcl 2 , 10 mm dithiothreitol and 1 mm atp supplemented with 0 . 5 μg of the following sali linker ## str3 ## with 1 unit of t4 dna ligase at 15 ° c . for 18 hours . the reaction mixture was used to transform e . coli w3110 , and a clone exhibiting ampicillin and tetracycline resistance ( ap r , tc r ) was obtained , a plasmid was isolated from the clone , and the plasmid was analyzed by restriction enzyme cleavage to confirm conversion of ecori site to sali site . the plasmid was designated as pbr322 - sali . five μg of the plasmid pbr322 - sali was partially digested with 8 units of sali in 50 μ1 of sali buffer comprising 6 mm tris - hcl ( ph 7 . 9 ), 150 mm nacl and 6 mm mgcl 2 at 37 ° c . for 60 minutes , and then digested completely with 24 units of bamhi . the reaction mixture was subjected to agarose gel electrophoresis to recover the second large dna fragment containing a promoter region of tetracycline resistance gene ( no . 1 fragment in fig1 ). on the other hand , 5 μg of plasmid pαne2 was digested completely with 24 units of bamhi and 24 units of ecori in 100 μl of sali buffer at 37 ° c . for 60 minutes , and the reaction mixture was subjected to agarose gel electrophoresis to recover the largest dna fragment containing β - gal 1007 gene expressed under the lac promotor and ampicillin resistance gene ( no . 2 fragment in fig1 ). the plasmid pαne2 was described in detail in japanese unexamined patent publication no . 58 - 63395 , and e . coli wa 802 / pαne2 was designated as sbm 102 , and deposited with the fri as ferm p - 6031 on june 19 , 1981 . moreover , 5 μg of plasmid ps224 - 3 containing γ - hanp gene the 3 &# 39 ;- terminal part of which corresponds to an α - hanp gene , described in japanese unexamined patent publication no . 60 - 262592 , european patent publication no . 0164273 , was digested completely with 24 units of pvuii and 80 units of sali in 100 μl of sali buffer at 37 ° c . for 60 minutes . the reaction mixture was then subjected to agarose gel electrophoresis to recover the smallest dna fragment containing the α - hanp gene lacking the n - terminal portion ( no . 3 fragment in fig1 ). on the other hand , a dna fragment of the following structure : ## str4 ## was synthesized . the dna fragment contains a codon for lysine residue as a linker amino acid and a portion of α - hanp gene which supplements the above - mentioned portion lacking in the no . 3 fragment , and has ecori site and pvuii site on both terminals respectively . the dna fragment is that of no . 4 in fig1 . the above - mentioned four dna fragments were ligated by the same procedure as described above , and the ligation mixture was used to transform e . coli w3110 , and clones exhibiting ampicillin and tetracycline resistance were obtained . from the clones plasmids were isolated and analyzed by restriction enzyme cleavage to obtain a desired plasmid pghα201 . five μg of the plasmid pghα20l was digested completely with 24 units of drai in 50 μl of sali buffer at 37 ° c . for 60 minutes , and the largest dna fragment was recovered by agarose gel electrophoresis . the dna fragment was ligated according to the same procedure as described above , and the ligation mixture was used to transform e . coli w3110 , and ampicillin sensitive and tetracycline resistant clones were obtained . plasmids from the clones were analyzed by restriction enzyme cleavage to select a desired plasmid pghα1007 . e . coli w3110 / pghα1007 containing the plasmid pghα1007 was designated as sbm 284 and deposited with the fri as ferm p - 8728 on apr . 3 , 1986 , and transferred to deposition under the budapest treaty as ferm bp - 1748 on feb . 22 , 1988 . reference example 2 . construction of plasmids pghα650 , pghα439 and pghα210 ( decrease of size of βgal gene ) ( fig1 ) five μg of the plasmid pghα1007 was digested completely with 8 units of ecori and 24 units of saci in 50 μl of ta buffer at 37 ° c . for 60 minutes . the reaction mixture was subjected to agarose gel electrophoresis to recover the largest dna fragment . the ecori and saci ends of the dna fragment were converted to blunt ends according to the same procedure as described above , and the dna fragment was self - ligated . the ligation mixture was then used to transform e . coli w3110 to obtain tetracycline resistant clones . plasmids were isolated from the clones according to a conventional process by restriction enzyme cleavage to obtain a desired plasmid pghα650 . plasmid pghα439 was constructed according to the same procedure as described above for the construction of plasmid pghα650 except that hindiii buffer was used in place of the ta buffer , and 24 units of mlui was used in place of saci . moreover , plasmid pghα210 was constructed according to the same procedure as described above for the construction of pghα650 except that hindiii buffer was used in place of ta buffer , and 24 units of aatii was used in place of saci . the plasmid pghα650 contains dna fragment coding for a fusion protein designated as βgal 650αhanp wherein the c - terminal of a polypeptide corresponding to 650 amino acid residues of an n - terminal of a βgal protein as partner protein was linked with an n - terminal of α - hanp as the target peptide via two additional amino acid residues glu - phe and a linker amino acid lysine . the plasmid pghα439 has a similar structure to that of pghα650 , except that pghα439 contains a dna fragment coding for a fusion protein βgal439αhanp wherein the partner protein corresponds to the n - terminal of a β - gal protein consisting of 439 amino acid residues . the plasmid pghα210 has a similar structure to that of pghα650 except that pghα2l0 contains dna fragment coding for a fusion protein βgal210αhanp wherein the partner protein corresponds to the n - terminal of a β - gal protein consisting of 210 amino acid residues . five μg of pbr322 was digested completely with 10 units of bali in 50 μ1 of bali buffer comprising 10 mm tris - hcl ( ph 7 . 6 ) and 12 mm mgcl 2 at 37 ° c . for 60 minutes , and the digested dna was recovered by ethanol precipitation . next , the recovered dna was dissolved in 50 μl of sali buffer , and digested completely with 24 units of pvuii , and the reaction mixture was subjected to agarose gel electrophoresis to recover the largest dna fragment having blunt ends . the dna fragment was then self - ligated according to the same procedure as described above , and the reaction mixture was used to transform e . coli w3110 . plasmids were obtained from tetracycline and ampicillin resistant clones , and plasmid lacking the pvuii - bali dna fragment of 622 bp designated as pbr322δbali was obtained . five μg of pbr322δbali was digested completely with 24 units of drai and 24 units of ecorv in 50 μl of sali buffer , at 37 ° c . for 60 minutes and the largest dna fragment containing an origin of replication was recovered by agarose gel electrophoresis . on the other hand , 5 μg of the plasmid pghα210 constructed in reference example 2 was digested completely with 24 units of ecorv at 37 ° c . for 60 minutes , and the second largest dna fragment containing gene coding for the fusion protein βgal1210αhanp was recovered by agarose gel electrophoresis . these two dna fragments were ligated according to the same procedure as described above , and the reaction mixture was used to transform e . coli w3110 to obtain tetracycline resistant clones . plasmids obtained from the clones were analyzed according to a conventional manner , and a desired clone e . coli w3110 / pghα210rop - was obtained . this plasmid pghα210rop - lacks a function of a region regulating the replication of plasmid ( called &# 34 ; rop &# 34 ;). according to the same procedure as described above , but starting with the plasmid pghα439 , plasmid pghα439rop - lacking a function of rop was constructed . after preparing transformants e . coli w3110 / pghα439rop - transformed with pghα439rop - and e . coli w3110 / pghα210rop - transformed with pghα210rop - , amounts of the production of fusion proteins βgal439αanp and βgal1210αhanp were measured by sds page , and the result was compared with those obtained for plasmids pghα439 and pghα210 . as a result , it was found that especially in the case of pghα210 , lack of the rop function greatly increased the production of the fusion protein . note , although when the production of fusion protein was low , the addition of an inducer iptg was highly effective , and where the number of copies increased due to the lack of the rop function , the addition of iptg was less effective . plasmid pp l lacz &# 39 ; 210αhanp wherein lac promoter in pghα210 was replaced by a p l promoter of the λ phage was constructed . according to the same procedure as described by morinaga , y . biotechnology 2 , 636 , 1984 , an ecori site was inserted into pghα210 immediately upstream of the lacz &# 39 ; gene to construct a plasmid pghα210 - ecori . namely , 5 μg of pghα210 was digested to completion with 24 units of hpai in 50 μl of ta buffer at 37 ° c . for 60 minutes , and the largest dna fragment containing α - hanp structural gene and tetracyclic resistance gene ( tc r ) ( no . 1 fragment in fig1 ) was electrophoretically separated and recovered . next , the dna fragment was treated with alkaline phosphatase in 50 μl of 100 mm tris - hcl ( ph 8 . 0 ) buffer to eliminate phosphate at the 5 &# 39 ; ends . next , 5 μg of pghα210 was digested to completion with 80 units of sali in 50 μl of sali buffer at 37 ° c . for 60 minutes , and the largest dna fragment lacking a part of the tetracycline resistant gene ( no . 2 fragment in fig1 ) was electrophoretically separated and recovered . on the other hand , a single stranded oligonucleotide having the following sequence : and having ecori site and phosphorylated at the 5 &# 39 ; end thereof ( no . 3 fragment and having ecori site in fig1 ) was chemically synthesized . two μg each of the double stranded dna fragments prepared as above and 1 μg of the above - mentioned chemically synthesized single stranded dna fragment was mixed in 36 μl of p / l buffer comprising 6 mm tris - hcl ( ph7 . 5 ), 50 mm nacl , 8 mm mgcl 2 and 1 mm β - mercaptoethanol , and the mixture was heated to 95 ° c . to denaturate the double stranded dna fragment to convert to a single stranded dna fragment , and gradually cooled to 30 ° c . over 60 minutes to anneal these dna fragments . to the reaction mixture were added 1 μl of 25 mm dntps and 2 units of klenow fragment of dna polymerase , as well as 2 μl of 10 mm atp and 2 units of t4 dna ligase , and a reaction was carried out to complete a double stranded circular dna . the reaction mixture was used to transform e . coli w3110 , and tetracycline resistant clones were obtained . plasmids from the clones were analyzed in a conventional manner to obtain a desired plasmid pghα210 - ecori containing a newly added ecori immediately upstream of lacz &# 39 ; gene . five μg of the above - mentioned plasmid pghα210ecori was partially digested with 0 . 2 units of ecori in 50 μl of hindiii buffer at 37 ° c . for 60 minutes , and the linear dna fragment was electrophoretically separated and recovered . next , the recovered dna fragment was digested completely with 80 units of sali in 100 μl of sali buffer at 37 ° c . for 60 minutes , and the second largest dna fragment containing a structural gene for βgal210αhanp ( no . 1 fragment in fig1 ) was electrophoretically separated and recovered . on the other hand , 5 μg of the plasmid ps224 - 3 described above was digested completely with 16 units of ecori and 24 units of avai in 50 μl of hindiii buffer at 37 ° c . for 60 minutes , and the largest dna fragment containing the p l promoter of the λ phage and an ampicillin resistance gene ( no . 2 fragment in fig1 ) was electrophoretically separated and recovered . a double stranded dna fragment having the following sequence : ## str5 ## and having a sali cohesive end and an avai cohesive end ( no . 3 fragment in fig1 ) were prepared by chemical synthesis . these three dna fragments were ligated according to the same procedure as described above , and the ligation mixture was used to transform e . coli w3110 / c 1 , and ampicillin and kanamycin resistant clones were obtained . plasmids from the clones were analyzed in a conventional manner to obtain a desired plasmid pp l lacz &# 39 ; 210αhanp . reference example 5 . construction of plasmid pghαbal having shortened β - gal region ( fig1 ) ten μg of the plasmid pghα439rop - constructed in reference example 3 was digested completely with 100 units of aatii in 100 μl of hindiii buffer at 37 ° c . for 60 minutes , and the digested dna was recovered by ethanol precipitation . next , the recovered dna was dissolved in 100 μl of bal 31 buffer comprising 20 mm tris - hcl ( ph 8 . 0 ), 12 mm cacl 2 , 12 mm mgcl 2 , 0 . 2 m nacl , and 1 mm edta , and treated with 10 units of bal 31 . after 5 , 10 , and 20 minutes , 30 μl of the reaction mixture was sampled and mixed with a same volume of phenol / chloroform 1 : 1 to terminate the reaction . after eliminating the phenol layer ( lower layer ), the aqueous layer was extracted with ethyl ether to eliminate the remaining phenol , and dna was recovered by ethanol precipitation . next , the dna fragment from each sample was dissolved in 30 μ l of ta buffer , and digested completely with 15 units of ecori , and 10 μl of the reaction mixture was subjected to 2 % agarose gel electrophoresis . to the remaining 20 μl samples from the 10 minute reaction and 20 minute reaction , which provided a dna fragment smaller than 400 bp , were added 70 μl of ta buffer , 2 μl of 25 mm dntps , 5 μl of 100 mm dithiothreitol and 4 units of t4 dpase , and the reaction was carried out at 37 ° c . for 30 minutes to convert cohesive ends of the dna fragment to blunt ends . after collection of each dna fragment by ethanol precipitation , the dna fragment was self - ligated with t4 dna ligase according to the same procedure as described above , and the ligation mixture was used to transform e . coli w3110 , and 54 e . coli w3110 / pghαbal clones were obtained . among the plasmids from the 54 clones , the plasmid pghαbal 43 was used for further experiments . plasmid pin4gif54 was constructed , as shown in fig1 , from ( 1 ) dna fragment containing the lipoprotein gene promoter region ( indicated by lpp in the figure ) as obtained by digestion of the plasmid pinia2 with the restriction enzymes xbai and psti , ( 2 ) oligonucleotide having xbai and ecori cohesive ends and ( 3 ) dna fragment containing the hinf - γ gene as obtained by digestion of the plasmid pgif54 with ecori and psti . the procedure followed was as described hereinbelow . the restriction enzymes used were all products of takara shuzo kk . the plasmid pini - a2 is a gift from dr . inoue of new york state university . a host escherichia coli strain obtained by transformation with said plasmid has been named ja22l / pini - a2 and deposited with the fermentation research institute , 1 - 3 , higashi 1 - chome , tsukuba - shi , ibaraki , japan , under deposit no . ferm bp - 320 , on july 18 , 1983 under the budapest treaty . the pini - a2 dna ( 3 μg ) was digested with 15 units each of xbai and psti in 150 μl of 1 × ta solution ( 33 mm tris acetate buffer ph 7 . 6 , 66 mm potassium acetate , 10 mm magnesium acetate and 0 . 5 mm dithiothreitol ) at 37 ° c . for 60 minutes . the reaction mixture was subjected to 1 . 0 % agarose gel electrophoresis and a gel portion located at the position corresponding to about 980 b . p . ( base pairs ) was cut out and placed in a dialysis tube , and the xbai - psti dna fragment was eluted by electrophoresis . after removal of ethidium bromide from the eluate by adding an equal amount of phenol thereto , 2 . 5 volumes of ethanol was added . after standing at - 80 ° c . for 30 minutes , the mixture was centrifuged at 10 , 000 rpm for 10 minutes , whereby the dna fragment was obtained as an ethanol precipitate . to this ethanol precipitate was added 10 μl of distilled water for dissolving the dna fragment . plasmid pgif54 is essentially the same plasmid as pgif4 disclosed in japanese patent application no . 86 , 180 / 1982 . an escherichia coli transformant , wa802 / pgif4 , obtained by transformation with said plasmid containing the chemically synthesized gene coding for the amino acid sequence of hifn - γ as shown in fig6 has been named sbmgl05 and deposited with the fermentation research institute , the agency of industrial science and technology as ferm p - 6522 on may 6 , 1982 , and transferred to deposition under the budapest treaty , as ferm bp - 282 , on may 2 , 1983 . the pgif54 dna ( 3 μg ) was digested with 15 units each of ecori and psti in 30 μl of 1 × ta solution at 37 ° c . for 60 minutes , followed by 0 . 7 % agarose gel electrophoresis , whereby an ecori - psti dna fragment of about 3 . 4 kb was eluted from the gel . the eluate was subjected to phenol treatment and ethanol precipitation in the same manner as above . to the ethanol precipitate , 10 μl of distilled water was added for dissolution of the dna fragment . for the expression of complete hinf - γ protein , an oligonucleotide having the shine - dalgarno ( sd ) sequence downstream from the xbai cleavage site of pinia2 and further having an ecori cohesive end , namely the oligonucleotide ## str6 ## was synthesized by the solid phase method . the synthetic procedure has been disclosed in detail in japanese patent application no . 86 , 180 / 1982 . the above oligonucleotide ( 100 picomoles ) was phosphorylated at the 5 &# 39 ;- oh in 30 μl of a kinase reaction solution ( 50 mm tris hydrochloride buffer , ph 8 . 0 , 10 mm mgcl 2 , 10 mm dithiothreitol ), with 2 units of t4 polynucleotide kinase ( takara shuzo kk ) added , at 37 ° c . for 60 minutes . the plasmid pin4gif54 was constructed by ligation of the three dna fragments prepared above in accordance with the following procedure . thus , to a mixture of 5 μl of a solution of the xbai - psti dna fragment of pinia2 ( solution of the ethanol precipitate in 10 μl of distilled water ), 5 μl of a solution of the ecori - psti dna fragment of pgif54 ( solution of the ethanol precipitate in 10 μl of distilled water ) and 3 μl of a solution of the phosphorylated oligonucleotide ( 10 picomoles ), there were added 2 μl of a ligation reaction medium 10 - fold higher in concentration ( 20 mm tris hydrochloride buffer , ph 7 . 6 , 10 mm mgcl 2 ), 2 μl of 4 mm atp and 1 μl of t4 dna ligase ( boehringer mannheim ) ( 5 units ), and the ligation was carried out at 16 ° c . overnight . escherichia coli wa802 was cultured in 2 . 0 ml of l - broth at 37 ° c . overnight , 0 . 3 ml of the culture broth was added to 30 ml of l - broth , and the culture was shaken at 37 ° c . for 2 hours , followed by centrifugation at 3 , 000 rpm for 10 minutes . to the thus - obtained cells was added 10 ml of 50 mm cacl 2 for suspending the cells , and centrifugation was conducted at 3 , 000 rpm for 10 minutes . to the thus - obtained cells was added 1 . 0 ml of 50 mm cacl 2 solution , and the mixture was allowed to stand in an ice bath for 60 minutes . to 0 . 2 ml of this suspension of ca ++ - treated cells was added 10 μl of the ligation reaction mixture obtained in example i - d ( containing the above - mentioned three dna fragments ligated ), the mixture was allowed to stand in an ice bath for 60 minutes , than 2 ml of l - broth was added and incubation was conducted at 37 ° c . for 60 minutes . the culture broth was used for plating on nutrient agar medium ( bbl ) containing 40 μg / ml of ampicillin . after incubation at 37 ° c . overnight , ampicillin - resistant transformants were selected . one of the transformants obtained was used for plasmid dna separation therefrom by the conventional method ( cleared lysate method ). the base sequence of the dna at and around the xbai - ecori region inserted was determined by the maxam - gilbert method ( methods in enzymology , 65 : 499 - 560 , 1980 ) and it was confirmed that the dna had the desired dna base sequence . this plasmid was named pin4gif54 and the transformant escherichia coli strain carrying the same was named wa802 / pin4gif54 . the oligonucleotide having the sd sequence aggaggt and xbai and ecori cohesive ends at the 5 &# 39 ;- ends , namely ## str7 ## was synthesized by the solid phase method mentioned above ( cf . japanese patent application no . 86 , 180 / 1982 ). the above oligonucleotide ( 100 picomoles ) was phosphorylated at the 5 &# 39 ;-- oh , at 37 ° c . for 60 minutes , in 50 μl of the kinase reaction solution with 2 units of t4 polynucleotide kinase ( takara shuzo ) added , as mentioned above . pin4gif54 ( 2 . 5 μg ) was digested with 5 units each of xbai and ecori in 30 μl of 1 × ta solution at 37 ° c . for 60 minutes for cleaving the dna . after cleavage , 0 . 7 % agarose gel electrophoresis was carried out and an xbai - ecori dna fragment of about 4 . 3 kb ( sd sequence - free longer fragment ) was eluted from the gel by electrophoresis as mentioned above . the eluate was subjected to phenol treatment and ethanol precipitation as mentioned above and , to the ethanol precipitate , 10 μl of distilled water was added for dissolution of the dna fragment . the plasmid pin5gif54 was constructed by ligating the above two dna fragments in the following manner . thus , to 5 μl a solution of the phosphorylated oligonucleotide ( 10 picomoles ), there were added 3 μl of the ligation reaction solution 10 - fold in concentration ( mentioned hereinbefore ), 10 μl of 100 mm dtt and 4 mm atp in distilled water , and 1 μl ( 5 units ) of t4 dna ligase ( boehringer mannheim ). the mixture was incubated at 16 ° c . overnight . in the same manner as above ( example ii - a ), cells of escherichia coli wa802 as grown in l - broth were treated with cacl 2 , and 0 . 2 ml of cell suspension was mixed with the ligation reaction mixture obtained in example iv - c for effecting transformation of escherichia coli wa802 . transformant selection was conducted using a nutrient agar medium ( bbl ) containing 40 μg / ml of ampicillin . using one of the transformants thus obtained , plasmid separation was performed by the conventional method ( cf . example ii - a ), and the dna base sequence at and around the insert region , namely xbai - ecori region , was analyzed . as shown in fig5 pin5gif54 must be free of the xbai cleavage site originally present in pin4gif54 ( fig3 ). therefore , the plasmid separated was treated with xbai , followed by 0 . 7 % agarose gel electrophoresis and determination , for the plasmid dna remaining uncleaved with xbai , of the dna sequence at and around the oligonucleotide fragment insert by the maxam - gilbert method . the results obtained confirmed the presence of the intended dna base sequence . this plasmid was named pin5gif54 , and the wa802 strain transformed therewith was named wa802 / pin5gif54 . a portion ( 5 μg ) of pin5gif54 was completely digested with 20 units of aatii and 20 units of sali . the cohesive aatii end ( 3 &# 39 ; terminus ) and sali end ( 5 &# 39 ; terminus ) were made blunt using t4 dna polymerase and 4dntp ( including datp , dgtp , dctp and dttp ); the aatii end was subjected to trimming whereas the sali end was subjected to filling - in . the digested plasmid was separated by agarose gel electrophoresis and a dna fragment ( ca . 750 bp ) containing the lipoprotein promoter ( lppp ) and gif gene was electro - eluted from the gel by electrophoresis . dna recovery was subsequently made by precipitation with ethanol . a portion ( 5 μg ) of pbr322 was completely digested with 20 units of ecori and the resulting sticky end was made blunt by the filling - in technique described above . the plasmid was then digested completely with 20 units of ahaiii . the digested plasmid was separated by agarose electrophoresis and a dna fragment ( ca . 3 . 3 kd ) containing the dna replication initiation origin and tetracycline - resistance gene was obtained by the method described above . the two dna fragments were mixed , and the mixture was subjected to ligation at 15 ° c . for 18 hours using t4 dna ligase ( 1 unit ) in a ligation solution ( 20 μl ) composed of 20 mm tris - hcl ( ph 7 . 4 ), 10 mm mgcl 2 , 10 mm dtt ( dithiothreitol ), and 1 mm atp . after treatment with 0 . 3 ml cacl 2 , e . coli w3ll0 was transformed by addition of 10 μl of the ligation solution . tetracycline resistant clones of the transformants were analyzed routinely to obtain w3ll0 clones having pin5t4 . example 1 . increase of recovery of α - hanp by hydrolysis of βgal210αhanp under dtt reduction as described in reference example 2 , βgal210αhanp is a fusion protein comprising α - hanp and a polypeptide having 210 amino acid residues of β - galactosidase . e . coli w3110 / pghα210rop - was cultured in a medium containing 0 . 5 % glycerol , 2 . 4 % yeast extract , 1 . 2 % trypton ( trademark for difco ; a product prepared by the partial hydrolysis of casein using typsin ) and 100 mm potassium - phosphate ( ph 7 . 5 ), and supplemented with tetracycline at 37 for 14 hours . next , the bacterial cells were harvested and suspended in 10 mm tris - hcl buffer ( ph 9 . 3 ), and disrupted by ultrasonication . the disrupted product was centrifuged at 10 , 000 g for one minute to obtain a precipitate . the precipitate was washed with the same buffer , and solubilized in a same buffer containing 5m urea . an aliquot of the mixture was subjected to sds polyacrylamide gel electrophoresis ( sds - page ) to determine an amount of fusion protein produced . the remaining reaction mixture was hydrolyzed with api ( achromobacter protease i ; wako junyaku , japan ) and an aliquot of the reaction mixture was subjected to sds - page to confirm completion of the hydrolysis . the reaction mixture was then analyzed by hplc using ymc - a - 302ods column to determine the amount of liberated α - hanp . the amount of 3 - hanp estimated from hplc was about one fourth of the amount estimated from sds - page , suggesting that α - hanp bonded to a partner protein βgal 210 in any manner cannot be liberated . as the reason for this phenomenon , it may be suggested that at least one of three cysteine residues present with βgal 210 protein consisting of 210 amino acid residues derived from β - galactosidase and at least one of two cysteine residues present in α - hanp form disulfide bond ( s ), and therefore , α - hanp molecule cut off from βgal 210 molecule at a peptide bond adjacent to a linker amino acid is bonded to the βgal 210 molecule via the disulfide bond ( s ). b . increase of recovery of α - hanp from hydrolyzed βgal210αhanp by dtt reduction to determine the involvement of disulfide bonds , βgal210αhanp preparation hydrolyzed with api was reduced with dtt and amount of α - hanp was measured by hplc . as a result , as shown in table 1 , the amount of liberated α - hanp was increased four times in comparison with the amount thereof before the reduction with dtt . the amount of liberated α - hanp after the dtt reduction is approximately the same as an amount approximated from the result of sds - page . from the above - mentioned result , it is suggested that the low recovery of α - hanp from βgal210αhanp after api hydrolysis is due to the presence of the disulfide bonds between βgal 210 molecule and α - hanp molecule . example 2 . conversion of cysteine residue to serine residue in βgal 210 to confirm the involvement of disulfide bonds in the low recovery of α - hanp from βgal210αhanp after api hydrolysis , and to improve native βgal 210 as a partner in a fusion protein , all cysteine residues present in native βgal 210 were replaced by serine residues . the modified βgal 210 protein wherein all cysteine residues are replaced by serine residues is designated as βgal2l0 ( ser ); and a fusion protein comprising α - hanp and the βgal210 ( ser ) is designated as βgal210 ( ser ) αhanp . βgal 210 protein contains three cysteine residues at the positions 76 , 122 and 154 calculated from the n - terminal . plasmid pghα210 ( ser ) rop - containing a gene coding for a fusion protein comprising α - hanp and modified βgal 210 wherein the above - mentioned three cysteine residues are converted to serine residues . the cys → ser conversion provides restriction enzyme cleavage sites bglii , bamhi and avaii . three μg of the plasmid pghα210rop - constructed in reference example 3 was digested completely with 20 units of pvui and 12 units of ecori in 50 μl of h buffer comprising 10 mm tris - hcl ( ph 8 . 0 ), 7 mm mgcl 2 and 100 mm nacl at 37 ° c . for 60 minutes , and the largest dna fragment containing tetracycline gene ( no . 1 fragment in fig1 ) was electrophoretically separated and recovered . in the same manner , 3 μg of pghα210rop - was digested completely with 20 units of avai and 20 units of bamhi in 50 μl of h buffer at 37 ° c . for 60 minutes , and the largest dna fragment containing lacz &# 39 ; 210 - αhanp gene ( no . 2 fragment in fig1 ) was electrophoretically separated and recovered . on the other hand , three oligonucleotide primers having the following sequences : ## str8 ## were chemically synthesized . each oligonucleotide contains a codon for serine in place of a codon for cysteine , and a restriction enzyme cleavage site bglii , bamhi or avaii , and have been phosphorylated at the 5 &# 39 ;- ends thereof . two μg each of the above - prepared double stranded dna fragments and 1 μg each of the three oligonucleotide primers were mixed in 30μl of buffer p / l buffer comprising 6 mm tris - hcl ( ph7 . 5 ), 50 mm nacl , 8 mm mscl 2 and 1 mm β - mercaptoethanol , and the mixture was heated to 95 ° c . to denature the double stranded dna fragments to form single stranded dna fragment , and gradually cooled to 30 ° c . for 60 minutes to anneal the dna fragments . to the reaction mixture were added 1 μg of dntps and 2 units of klenow fragment of dna polymerase , as well as 2 units of t4 dna ligase and 2 μl of 10 mm atp to complete a circular double stranded dna . the reaction mixture was used to transform e . coli w3110 to obtain tetracycline resistant clones . plasmids from the clones were analyzed in a conventional manner to obtain a desired plasmid pghα210 ( ser ) rop - containing newly introduced restriction sites bglii , bamhi and avaii . e . coli w3110 / pghα210 ( ser ) rop - was cultured in a medium containing 0 . 5 % glycerol , 2 . 4 % yeast extract , 1 . 2 % trypton and 100 mm potassium - phosphate ( ph 7 . 5 ), and supplemented with tetracycline at 37 for 14 hours . next , the bacterial cells were harvested and suspended in 10 mm tris - hcl buffer ( ph 9 . 3 ), and disrupted by ultrasonication . the disrupted product was centrifuged at 10 , 000 g for 5 minutes to obtain a precipitate . the precipitate was washed with the same buffer , and solubilized in a same buffer containing 5 m urea . an aliquot of the mixture was subjected to sds - polyacrylamide gel electrophoresis ( sds - page ) to determine an amount of fusion protein produced . the remaining reaction mixture was hydrolyzed with api ( achromobacter protease i ; wako junyaku , japan ) and an aliquot of the reaction mixture was subjected to sds - page to confirm completion of the hydrolysis . the reaction mixture was then analyzed by hplc using ymc - a - 302ods column to determine the amount of liberated α - hanp . the amount of liberated α - hanp obtained from the result of hplc was roughly the same as that estimated from a result of sds - page , and dtt - reduction of api - hydrolyzed sample did not increase the amount of α - hanp liberated , as shown in table 1 . this result demonstrates that lower recovery of α - hanp from βgal210αhanp is due to the presence of cysteine residues in βgal 210 protein . moreover the above - mentioned result shows that α - hanp can be easily isolated from the fusion protein βgal210 ( ser ) αhanp without dtt - reduction and , therefore , without subsequent oxidation . table 1______________________________________ amount of α - hanp liberatedfusion protein - dtt + dtt______________________________________βgal210αhanp 6 . 7 mg 27 . 3 mg ( 0 . 25 ) ( 1 . 00 ) βgal210 ( ser ) αhanp 24 . 2 mg 25 . 0 mg ( 0 . 97 ) ( 1 . 00 ) ______________________________________ a process for the production of α - hanp through a fusion protein βgal210 ( ser ) αhanp has advantages , in addition to that ( 1 ) α - hanp can be produced in a large amount and ( 2 ) since expressed fusion protein is transferred to a precipitate while impurity proteins remain in a supernatant , the fusion protein is easily purified , as in use of βgal210αhanp , that ( 3 ) since βgal210 ( ser ) does not contain cysteine residue and , therefore , a disulfide bond is not formed between α - hanp and βgal210 ( ser ), α - hanp can be almost completely recovered from api - hydrolyzed fusion protein without dtt - reduction and subsequent oxidation . in industrial production of α - hanp , however , there are some problems . namely , ( a ) since the fusion protein βgal210 ( ser ) αhanp has a low solubility in an urea aqueous solution , a low concentration of the fusion protein , i . e ., high dilution of a reaction mixture is required in the api - hydrolysis process , and ( b ) since the efficiency of api - hydrolysis on βgal210 ( ser ) αhanp is rather low , a large amount of api enzyme per substrate and long reaction time is necessary , with a possibility of an undesirable modification of the target product α - hanp . taking into account the above - mentioned situation , to resolve the problems ( a ) and ( b ) while maintaining the advantages ( 1 ), ( 2 ), and ( 3 ) the present inventors attempted to shorten the size of a partner protein . for this purpose , a plasmid containing a dna coding for a fusion protein comprising α - hanp and a partner peptide of 97 amino acid derivative corresponding to the n - terminal of a β - galactosidase protein wherein 76th cysteine is replaced by serine was constructed . the peptide consisting of 97 amino acid residues of β - galactosidase is designated as βgal 97 ; and the fusion protein comprising α - hanp and the βgal 97 is designated as βgal97αhanp . moreover , the peptide consisting of 97 amino acid residues of β - galactosidase wherein 76th cysteine is replaced by serine is designated βgal97 ( ser ); and the fusion protein comprising α - hanp and the βgal97 ( ser ) is designated βgal97 ( ser ) αhanp . plasmid pghα97 ( ser ) rop - containing a gene for βgal97 ( ser ) αhanp wherein 76th amino acid cysteine has been replaced by serine in the fusion protein βgal97αhanp was constructed . ten μg of the plasmid pghαbal43 constructed in reference example 5 was digested completely with 50 units of psti and 50 units of ddei in 70 μl of h buffer at 37 ° c . for 60 minutes , and the digestion product was subjected to agarose gel electrophoresis to separate and recover a 283 bp dna fragment containing α - hanp gene ( no . 1 fragment in fig2 ). on the other hand , 3 μg of the plasmid pghα210 ( ser ) rop - constructed in example 1 was digested completely with 20 units of pvui and 20 units of psti in 50 μl of h buffer at 37 ° c . for 60 minutes , and the largest dna fragment containing tetracycline resistant gene ( no . 2 fragment in fig2 ) was electrophoretically separated and recovered . moreover , 10 μg of pghα210 ( ser ) rop - was digested completely with 50 units of pvui and 50 units of ddei in 70 μl of h buffer , and a 101 bp dna fragment containing a part of lacz &# 39 ; gene ( no . 3 fragment in fig2 ) was separated and recovered by agarose gel electrophoresis . these three dna fragments were ligated in 20 μl of ligation mixture comprising 20 mm tris - hcl ( ph7 . 4 ), 10 mm mgcl 2 and 10 mm dithiothreitol using 1 unit of t4 dna ligase at 15 ° c . for 18 hours , and the reaction mixture was used to transform e . coli w3ll0 to obtain tetracycline resistant clones . plasmids from the clones were analyzed in a conventional manner to select a desired plasmid pghα97 ( ser ) rop - containing lacz &# 39 ; gene coding for fusion protein βgal97 ( ser ) αhanp wherein 76th cysteine has been replaced by serine . escherichia coli sbm288 containing the plasmid pghα97 ( ser ) rop - was deposited with the fri under the budapest treaty as ferm bp - 1253 on jan . 9 , 1987 . when e . coli w3110 / pghα97 ( ser ) rop - , i . e ., e . coli w3110 transformed with plasmid pghα97 ( ser ) rop - was cultured in a medium containing 0 . 5 % glycerol , 2 . 4 % yeast extract , 1 . 2 % trypton and 100 mm potassium - h - phosphate ( ph 7 . 5 ) and supplemented with tetracycline at 37 ° c . for 14 , the productivity of βgal97 ( ser ) αhanp was lower than that of βgal97αhanp . therefore , to improve the productivity of βgal97 ( ser ) αhanp , an attenuator terminator of tryptophan operon ( trp a ) was inserted immediately downstream of the α - hanp structural gene to construct plasmid pghα97s . ten μg of the plasmid pghα97 ( ser ) rop - was digested completely with 50 units of bglii and 50 units of rsai in 100 μl of h buffer at 37 ° c . for 60 minutes , and a 154 bp dna fragment containing c - terminal region of βgal97 ( ser ) gene and n - terminal region of α - hanp gene ( no . 1 fragment in fig3 ) was electrophoretically separated and recovered . next , 3 μg of pghα97 ( ser ) rop - was digested completely with 20 units of bglii and 40 units of sali in 50 μl of s buffer comprising 10 mm tris - hcl ( ph 8 . 0 ), 150 mm nacl and 7 mm mgcl 2 at 37 ° c . for 60 minutes , and the largest dna fragment containing an origin of replication ( no . 2 fragment in fig3 ) was electrophoretically separated and recovered . moreover , 3 μg of pbr322 was digested completely with 10 units of ecori and 40 units of sali in 50 μl of s buffer at 37 ° c . for 60 minutes , and a smaller dna fragment containing a promoter region of tetracycline resistant gene ( no . 3 fragment in fig3 ) was electrophoretically separated and recovered . on the other hand , a chemically synthesized double stranded dna fragment having the following sequence : ## str9 ## and having rsai blunt end and ecori cohesive end at its ends ( no . 4 fragment in fig3 ) was prepared . the above - prepared four dna fragments were ligated by the same procedure as described above , and the ligation mixture was used to transform e . coli w3110 . tetracycline resistant clones were obtained , and plasmids from the clones were analyzed in a conventional manner to obtain the desired plasmid pghα97s . e . coli w3110 / pghα97 ( ser ) rop - and e . coli w3110 / pghα97s , i . e ., e . coli w3110 transformed with pghα97s were cultured in a medium containing 0 . 5 % glycerol , 2 . 4 % yeast extract , 1 . 2 % trypton , 100 mm potassium - h - phosphate ( ph 7 . 5 ), supplemented with tetracycline at 37 ° c . for 14 hours , and amounts of produced βgal97 ( ser ) αhanp were compared by sds - page according to the same procedure as described in example 2 . b . for analysis of fusion protein . as seen from fig4 by introducing trp immediately downstream of α - hanp gene the productivity of βgal97 ( ser ) αhanp remarkably increased . amount of βgal97 ( ser ) αhanp produced by e . coli w3100 / pghα97s is approximately the same amount of βgal210 ( ser ) αhanp produced by e . coli w3110 / pghα210 ( ser ) rop - ; this means that the productivity of α - hanp per culture medium in the case of w3110pghα97s is about two times higher than that in the case of w3110 pghα210 ( ser ) rop - . the fusion protein βgal97 ( ser ) αhanp also , the same as βgal210αhanp andβga210 ( ser ) αhanp , transferred to a precipitation fraction during centrifugation of the cell disruptant , while e . coli proteins are transferred to a supernatant fraction ( see fig5 ). as seen from table 2 , although the amount of α - hanp liberated from api - hydrolyzed βgal97αhanp was increased by dtt - reduction as in the case of βgall210αhanp , the amount of αhanp liberated from api - hydrolyzed βgal97 ( ser ) αhanp was not increased by dtt - hydrolyzed reduction as in the case of βgal210 ( ser ) αhanp . table 2______________________________________ amount of α - hanp liberatedfusion protein - dtt + dtt______________________________________βgal97αhanp 7 . 8 mg 25 . 0 mg ( 0 . 31 ) ( 1 . 00 ) βgal97 ( ser ) αhanp 24 . 8 mg 30 . 9 mg ( 0 . 80 ) ( 1 . 00 ) ______________________________________ moreover , the solubility of the fusion protein βgal97 ( ser ) αhanp in a 5 m urea aqueous solution is about ten times higher than that of βgal210 ( ser ) αhanp . this means , taking into account the fact that the proportion of α - hanp in βgal97 ( ser ) αhanp is about two times higher than that in βgal210 ( ser ) αhanp , that concentration of α - hanp in βgal97 ( ser ) αhanp dissolved in 5 m urea is about 20 times higher than that in the case of βgal210 ( ser ) αhanp . the sensitivity of βgal97 ( ser ) αhanp to api - hydrolysis was about 100 times higher ( about 200 times for α - hanp per se .) than that of βgal210 ( ser ) αhanp . accordingly , in the next example , the reason for the high sensitivity of βgal97 ( ser ) αhanp to api - hydrolysis was studied . in βgal97 ( ser ) αhanp , βgal97 ( ser ) is linked with α - hanp via gln - phe - lys wherein gln is the 98th amino acid in βgal97 ( ser ) αhanp , and api hydrolyzes a peptide bond at the c - terminal side of lys . on the other hand , in βgal210 ( ser ) αhanp , βgal210 ( ser ) is linked with α - hanp via glu - phe - lys wherein glu is the 2llth amino acid in βgal210 ( ser ) αhanp . the present invention hypothesized that the reason why βgal97 ( ser ) αhanp is more sensitive to api - hydrolysis than βgal210 ( ser ) αhanp is because although the negative charge on σ carboxyl group of glu in βgal210 ( ser ) αhanp interferes with the positive charge on lys , σ carboxyamide of gln in βgal97 ( ser ) αhanp does not have a corresponding negative charge and , therefore , there is no interference with a corresponding positive charge on lys . to confirm the hypothesis , plasmid pghα97se containing a gene coding for a fusion protein βgalα97 ( ser ) αhanp wherein 97th amino acid gln in βgal97 ( ser ) αhanp has been replaced by glu ( that is , gln - phe - lys at junction part has been converted to glu - phe - lys ) was constructed , and the sensitivities of fusion proteins from plasmids pghα97s and pghα97se were compared . three μg of the plasmid pghα97s constructed in example 3 was digested completely with 20 units of bamhi and 20 units of avai in 50 μl of h buffer at 37 ° c . for 60 minutes , and the largest dna fragment containing α - hanp gene ( no . 1 fragment in fig6 ) was electrophoretically separated and recovered . moreover , 3 μg of pghα97s was digested completely with 20 units of bglii and 20 units of ecori in 50 μl of h buffer at 37 ° c . for 60 minutes , and the largest dna fragment containing tetracycline resistant gene ( no . 2 fragment in fig6 ) was electrophoretically separated and recovered . on the other hand , a single stranded oligonucleotide having the following sequence : ## str10 ## containing a codon for glutamic acid in place of a codon for glutamine , and phosphorylated at the 5 &# 39 ;- end thereof ( no . 3 fragment in fig6 ) was chemically synthesized . the above - prepared two double stranded dna fragments and the single stranded origonucleotide were mixed in p / l buffer , and the mixture was heated to 95 ° c . to denaturate the double stranded dna fragments to form single stranded dna fragments , and gradually cooled to 30 ° c . for 60 minutes to anneal the dna fragment to double stranded dna . to the reaction mixture , were added dntps and dna polymerase klenow fragment , as well as t4 dna ligase and atp , and reaction was carried out to form a closed circular . the reaction mixture was used to transform e . coli w3110 . tetracycline resistant clones were obtained , and plasmids from the clones were analyzed in a conventional manner to select a desirable plasmid pghα97se containing a gene coding for βgalα97 ( ser ) eαhanp wherein amino acid residues at junction region gln - phe - his has been converted to glu - phe - lys . e . coli w3100 / pghα97s and e . coli w3100 / pghα97es were cultured in a medium containing 0 . 5 % glycerol , 2 . 4 % yeast extract , 1 . 2 % trypton and 100 mm potassium - h - phosphate ( ph 7 . 5 ) supplemented with tetracycline at 37 ° c . for 14 hours . the cultured broth was centrifuged to harvest the bacterial cells which were then suspended in 10 mm tris - hcl ( ph 9 . 3 ), and disrupted by ultrasonication . the disrupted product was then centrifuged at 10000 g for one minute to obtain a precipitate , which was then washed with the same buffer . the washed precipitate was solubilized in the same buffer containing 5m urea , and the solubilized preparation was divided into four portions . to these preparations , 0 , 1 , 5 , and 25 ng / 5 μg αhanp of api were added respectively . each mixture was incubated at 30 ° c . for 45 minutes , and analyzed by sds - page . as seen from fig7 βgal97 ( ser ) eαhanp and βgal97 ( ser ) αhanp did not have different sensitivities to api - hydrolysis . therefore , the above - mentioned hypothesis was denied . probably , the high sensitivity of βgal97 ( ser ) αhanp to api - hydrolysis is due to the structure of βgal97 ( ser ) αhanp per se . plasmid pp l lacz &# 39 ; 97 ( ser ) αhanp was constructed by replacing the lac promoter in pghα97s by a p l promoter of the λ phage . ten μg of the plasmid pp l lacz &# 39 ; 210αhanp constructed in fig4 was digested completely with 50 units of ecori and 50 units of pvui in 100 μl of h buffer at 37 ° c . for 60 minutes , the reaction mixture was subjected to 5 % acrylamide gel electrophoresis to separate and purify a 143 bp dna fragment containing lacz &# 39 ; gene coding for n - terminal of β - galactosidase ( no . 1 fragment in fig8 ). moreover , 3 μg of pp l lacz &# 39 ; 210αhanp was digested completely with 20 units of ecori and 20 units of avai in 50 μl of h buffer at 37 ° c . for 60 minutes , and the largest dna fragment containing p l promoter and ampicillin resistant gene ( no . 2 fragment in fig8 ) was electrophoretically separated and purified . on the other hand , 3 μg of pghα97s was digested completely with 10 units of pvui and 10 units of avai in 50 μl of h buffer , and a dna fragment containing α - hanp gene and tetracycline resistant gene ( no . 3 fragment in fig8 ) was obtained . these three dna fragment were ligated by the same procedures described above , and the ligation mixture was used to transform e . coli w3110 / c 1 ( e . coli w3110 containing plasmid having c 1 875 gene and kanamycin resistant gene ). tetracycline , ampicillin and kanamycin resistant clones were obtained . plasmids from the clones were analyzed in a conventional manner to select desired plasmid pp l lacz &# 39 ; 97 ( ser ) αhanp . plasmid pinlacz &# 39 ; 97 ( ser ) αhanp was constructed by replacing p l promoter in plasmid pp l lacz &# 39 ; 97 ( ser ) αhanp by lpp promoter of e . coli outer membrane lipoprotein gene . ten μg of pp l lacz &# 39 ; 97 ( ser ) αhanp was digested completely with 50 units of bamhi in 100 μg of h buffer at 37 ° c . for 60 minutes , and then partially digested with 5 units of ecori , and the digestion product was subjected to 1 % agarose gel electrophoresis to separate and purify a 809 bp ecori - bamhi dna fragment containing βgal97 ( ser ) αhanp gene and a promotor region of tetracycline resistant gene ( no . 1 fragment in fig9 ). on the other hand , 3 μg of plasmid pin5t4 was digested completely with 20 units of ecori and 20 units of bamhi in 50 μl of h buffer at 37 ° c . for 60 minutes , and the largest dna fragment containing lpp promoter ( no . 2 fragment in fig9 ) was electrophoretically separated and purified . these two dna fragments were ligated by the same procedure as described above , and the ligation mixture was used to transform e . coli w3110 . tetracycline resistant clones were obtained , and plasmids from the clones were analyzed in a conventional manner to obtain desired plasmid pinlacz &# 39 ; 97 ( ser ) αhanp . e . coli w3110 / pgeα97s prepared in example 3 was cultured in 20 liter of a medium containing 0 . 4 % yeast extract , 0 . 4 % kh 2 po 4 , 0 . 4 % k 2 hpo 4 , 0 . 3 % na 2 hpo 4 , 0 . 02 % nh 4 cl , 0 . 12 % ( nh 4 ) 2 so 4 , 0 . 1 % mgso 4 . 7h 2 o , 1 . 5 % glucose , 2 % glycerine ( added 4 times ) ( ph 7 . 0 ) supplemented with tetracycline in a 30 liter jar fermenter , at 37 ° c . for 18 hours with aeration and agitation . the culture broth was subjected to a high pressure homogenization ( manton gaulin laboratory homogenizer l5m - 8ta ) at 8000 psi to disrupt the cells . the homogenate was centrifuged to obtain precipitate containing fusion protein , which was then washed with 10 mm tris - hcl ( ph 9 . 3 ) buffer . the washed precipitate was solubilized in 8 m urea solution . to the urea solution , 27 mm tris - hcl ( ph 9 . 3 ) was added to bring the urea concentration to 5 m . this urea solution was treated with 40 au of api ( wako junyaku , japan ) at 30 ° c . for 60 minutes , and the reaction mixture was filtered through a cartridge filter cwsc ( nippon milipore ltd .). next , the filtrate was chromatographed through zeta prep qae column ( nippon milipore ltd .) which adsorbs liberated βgal97 ( ser ), and intact βgal97 ( ser ) αhanp through βgal97 ( ser ) portion . a flow - through fraction containing liberated βgal97 ( ser ) was obtained , and this fraction was adjusted to ph 5 . 0 by adding acetic acid . next , the fraction was applied cm toyopearl 650m column equilibrated with 5 m urea in 10 mm ammonium formate ( ph 5 . 0 ). α - hanp was eluted stepwise with 225 mm nacl and 400 mm nacl to obtain a fraction containing α - hanp . the α - hanp fraction was applied to an spw - c - ods column ( chemco ) in medium performance liquid chromatography ( mplc ) ( murayama kagaku ) to desalt . finally α - hanp was purified by c 18 - hplc . the final α - hanp preparation provided a single peak in reverse hplc ( waters ) using ymc - a - 302ods column as shown in fig1 . amino acid composition of the α - hanp preparation thus obtained , as determined by an amino acid autoanalyzer ( hitachi seisakusho , 835 - 50 ) conformed to a value estimated from amino acid sequence of α - hanp . moreover , amino acid sequence of the above - mentioned α - hanp preparation as determined by a gas phase protein sequencer model 470a ( applied biosystem ) was the same as that of α - hanp . moreover the α - hanp preparation prepared by the present process was equivalent to a chemically synthesized α - hanp preparation ( peptide research ) in ( 1 ) natriuretic and blood pressure - lowering actions in sd rat , ( 2 ) relaxation activity on chick rectal tissue , and ( 3 ) cross - immunoreactivity in radioimmunoassay . according to the present process , about 5 g of fusion protein ( corresponding to about 1 g of α - hanp ) is obtained from 1 liter of culture broth , and recovery and purification yield is about 50 % to obtain α - hanp in a pure form . such a high productivity and high recovery are very advantageous in the industrial production of α - hanp . note , e . coli w3110 / c 1 / pp l lacz &# 39 ; 97 ( ser ) αhanp , containing plasmid pp l lacz &# 39 ; 97 ( ser ) αhanp constructed in example 5 , as well as e . coli w3110 / pinlacz &# 39 ; 97 ( ser ) αhanp , containing plasmid pinlacz &# 39 ; 97 ( ser ) αhanp constructed in example 6 , produced an amount of fusion protein βgal97 ( ser ) αhanp corresponding to about 30 % relating to the total proteins produced by the transformant , and the fusion protein isolated from the precipitation fraction . five μg of plasmid pghα97se was completely digested with 12 units of ecori and 80 units of sali in 100 μl of s buffer , and the largest dna fragment ( 1 ) was isolated and purified by agarose gel electrophoresis . on the other hand , 5 μg of plasmid pghα97se was completely digested with 80 units of sali in 100 μl of s buffer , and the second larget dna fragment ( 2 ) containing a promotor region of tetracyline resistance gene was isolated and purified by agarose gel electrophoresis . moreover , 1 μg of plasmid pucl8 ( takara shuzo ) was completely digested with 12 units of ecori and 80 units of sali in 100 μl of s buffer , and the reaction mixture was treated with phenol to inactivate the enzyme , and extracted with ethanol to eliminate the phenol and salts ( 3 ). the above - mentioned dna fragments ( 1 ), ( 2 ), and ( 3 ) were mixed and ligated , and the ligation mixture was used to transform e . coli w3110 , and tetracycline resistant clones were obtained . the clones were analyzed by a conventional procedure to select plasmid pg97sl8 wherein an ecori - sali dna fragment coding for the α - hanp gene in pghα97se is replaced by an ecori - sali dna fragment of a poly linker region in pucl8 having the following formula : ## str11 ## five μg of plasmid pg97sl8 was completely digested with 12 units of ecori and 12 units of bamhi in 50 μl of h buffer , and the largest dna fragment ( 4 ) was separated and purified by agarose gel electrophoresis . on the other hand , 5 μg of plasmid pgh97sl8 was completely digested with 12 units of βamhi in 50 μg of h buffer , and a dna fragment ( 7 ) containing a tetracycline gene promotor was separated and purified by agarose gel electrophoresis . moreover , 10 μg of plasmid pahpct38 was completely digested with 12 units of bamhi and 24 units of kpni in m buffer , and 81 bp kpni - bamhi dna fragment ( 6 ) coding for from the seventh tyr to 32nd pro calculated from the n - terminal of calcitonin , followed by gly - lys - lys - arg was separated and purified by polyacrylamide gel electrophoresis . on the other hand , a double stranded oligonucleotide ( 7 ) having the following sequence : ## str12 ## and having ecori cohesive end and kpni cohesive end was prepared . the above - mentioned dna fragments ( 4 ), ( 5 ), ( 6 ), and ( 7 ) were mixed and ligated , and the ligation mixture was used to transform e . coli w3110 to obtain tetracycline resistant clones . these clones were analysed by a conventional process to obtain a desired plasmid pg97shpct ( fig1 ). note , the above - mentioned plasmid pahpct38 is described in detail in japanese unexamined patent publication no . 58 - 203953 , and e . coli containing the plasmid pahpct38 , i . e ., escherichia coli e15 / pahpct38 designated as sbmcl38 was deposited with the fri as ferm p - 6523 on june 10 , 1982 , and transferred to international deposition under the budapest treaty as ferm bp - 282 on may 2 , 1983 . e . coli w3110 / pg97shpct transformed with the above - mentioned plasmid pg97shpct was cultured in a medium containing 0 . 5 % glycerine , 2 . 4 % yeast extreat , 1 . 2 % trypton and 100 mm potassium - phosphate ( ph 7 . 5 ) supplemented with tetracycline at 37 ° c . for 16 hours . next , the culture broth was centrifuged to obtain bacterial cells , which were then suspended in 10 mm tris - hcl ( ph 7 . 8 ) buffer and disrupted with a french pressure cells and press ( slm - aminco ) at 8000 psi . the disruptant was centrifuged at 1000 g for 10 minutes , to obtain both a supernatant and a precipitate , which were then analysed by sds - page . as a result , βgal97shpct was producted in approximately the same amount as βgal97sαhanp , and recovered in the precipitate fraction as in the case of βgal97sαhanp . this precipitate was suspended in 8 m aqueous urea solution , and to the suspension was added three volumes of 67 mm tris - hcl ( ph 7 . 8 ) to bring a concentration of urea to 2 m . to the diluted suspension was added protease v 8 ( boehringer mannheim ) in an amount of 1 / 2000 ( w / w ) relative to the substrate , and the whole was incubated at 37 ° c . for 10 minutes . an aliquot of the reaction mixture was analysed by reverse phase high performance liquid chromatography using ymc - a3020ds ( shimazu ). as a result , it was observed that , among ten glutamic acid residues present in βgal97shpct a bond at a glutamic acid residue present between βgal97s and hpct is most ready to be cleaved . moreover , the reaction mixture was subjected to column chromatography using zeta - prep qae equilibrated with 50 mm tris - hcl ( ph 7 . 8 ) buffer containing 2 m urea , and as a result , approximately 100 % of hpct was recovered in a flow - through fraction at a purity of about 95 %. this means that βgal97s is suitable as a partner protein not only for the productions of α - hanp but also for the production of human calcitanin precursor ( hpct ).