A METHOD OF SIMULTANEOUSLY PRODUCING A LARGE NUMBER OF Leu<17>-VIP-ANALOGS AND NEW Leu<17>-VIP-ANALOGS

Abstract: A method of simultaneously producing a large number of individual C-terminally extended analogs of Leu<17>-VIP is described. Said method comprises the steps of synthesizing two separate mixtures of equally long oligonucleotides coding for N-terminal and C-terminal portions of Leu<17>-VIP analogs, respectively, each having a few defined triplets which are made ambiguous and a few identical bases at the 3'-end, mixing said two mixtures to form a mixture of partially double stranded equally long DNA sequences, which are enzymatically converted into double stranded DNA sequences, subjecting the last mentioned mixture to cleavage, with two different enzymes to produce DNA sequences, which are inserted into similarly cleaved vectors by ligation, whereupon the vectors, are transformed into hosts, propagating said hosts to form colonies, which are analyzed one by one to establish the DNA sequences which code for a single protein, propagating separately under expression conditions those hosts which contain vectors having identified protein coding sequences, whereupon the separately expressed proteins are cleaved to release the C-terminally extended analogs of Leu<17>-VIP. Additionally there are disclosed new C-terminally extended analogs of Leu<17>-VIP and plasmids containing genes coding for the new analogs of Leu<17>-VIP.

Description: A method of simultaneously producing a large number of Leu 17-VIP-analogs and new Leu17-VIP-analogs . The present invention relates to a method of simultaneously producing a large number of peptide analogs and to new peptide analogs. The invention relates specifically to a method of simultaneously producing a group of individual C-terminally extended analogs of Leu 17-VIP (vasoactive intestinal polypep- tide), to new C-terminally extended analogs of Leu 17 -VIP and to plasmids containing genes having DNA sequen- ces which code for said new Leu 17-VIP analogs. Background The pharmaceutical industry, and independent research workers, are constantly searching for new compounds, which slightly differ from useful known compounds, with the aim of finding more potent, more specific etc derivatives. In case the known compound of interest is a peptide or polypeptide, one or several amino acid residues of the amino acid sequence thereof are substituted for other natural or unnatural amino acid residues. For screening purposes it would be desirable to be able to simultaneously produce a large number of analogs of a chosen peptide in order to save both time and money. The present invention provides a method of simul- taneously producing a large number of C-terminally extended analogs of Leu 17-VIP, wherein all amino acid residues are of L-configuration. Prior Art Vasoactive intestinal polypeptide (VIP) is a highly basic 28 amino acids long peptide with a C-ter¬ minal amide belonging to the glucagon-secretin family. VIP was first isolated by S. Said and V. Mutt in 1970 (Said, S.I. and Mutt, V. (1970) Science 169, 1217-1218) from porcine upper intestinal tissue, but has later been found both in nervous tissue and in endocrine cells (Said, S.I. (1982) Vasoactive Intestinal Peptide (Raven Press, N.Y.) and (Said, S.I. (1984) Peptides _5, 143-150). The biological effects of VIP include vasodilation of cerebral blood vessels, stimulation of prolactin release, stimulation of pancreatic exocrine secretion, effect on penile erection (Said, S.I. (1982) Vasoactive Intestinal Peptide (Raven Press, N.Y.) and (Rostene, W.H. (1984) Progress in Neurobiology 22, 103-129) and Mutt, V. (1983) in Brain Peptides, eds. Krieger, D.T., et al), potentiation of choliner- gically stimulated salivary flow (Lundberg, J.M. , et al (1982) Acta Physiol. Scand. 114, 329-337) and acting as surfactant in the lung (Barnes, P.J. (1987) TIPS, _8, 24-27) . Recently the precursor gene coding for human VIP has been isolated and sequenced (Bodner, M. , et al. (1985) Proc. Natl. Acad. Sci. USA, 8J2, 3548-3551) and (Linder, S., et al. (1987) Proc. Natl. Acad. Sci. USA, 8_4, 605-609). The sequence data show that at the 3 ' -end of the gene coding for VIP there are three additional triplets coding for the amino acid sequence Gly-Lys-Arg. It has been shown that C-terminal Gly-Lys- -Arg or Gly alone may act as substrate sites for C-ter¬ minal amidation by peptidylglycine -amidating -mono- oxygenase (PAMase) detected in various tissues (Bradbury, A.F., et al . (1982) Nature 298, 686-688) and (Eipper, B.A., et al (1983) Peptides _4, 921-928) and (Glembotski, C.C. τ et. al (1984) J. Biol. Chem. 259, 6385-6392) and (Gomez, S., et. al (1984) FEBS Lett. 167, 160-164) and (Eipper, B.A., et. al. (1985) Endocrinology, 116, 2497-2504) and Ouafik, H., et. al. (1987) Proc. Natl. Acad. Sci. USA, JJ4, 261-264). It is obvious that large scale production by genetic engineering methods of VIP, a peptide hormone of broad biological activity, and analogs thereof,. would be desirable. Although the human precursor gene for VIP has been isolated and characterized (Bodner, M. , et. al. (1985) Proc. Natl. Acad. Sci. USA, 8_2, 3548-3551) and Linder, S., et. al. (1987) Proc. Natl. Acad. Sci. USA, _8_4_, 605-609), its expression in another organism has not been reported yet. The C-termina.lly extended Leu 17-VIP analogs of the present invention have been produced in E. coli. Description of the invention In one aspect of the invention there is provided a method of simultaneously producing a group of individual C-terminally extended analogs of Leu -VIP (vasoactive intestinal polypeptide), said group consisting of equally long peptides having the amino acid sequence His-Ser-Asp-Ala-Val-Phe-X-Asp-Asn-Tyr-Thr-Arg-Leu-Y- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 -Lys-Gln-Leu-Ala-Val-Z-Lys-Tyr-Leu-Asn-Ser-Ile-Leu- 15 16 17 18 -19 20 21 22 23 24 25 26 27 -Asn-W 28 29 wherein X represents Thr, Ser, Pro or Ala, Y represents Thr, Lys, lie or Ala Z represents Thr, Lys, lie or Arg, and W represents Gly or Gly-Lys-Arg, 17 and of equally long peptides which are also Leu -VIP- -W 29 analogs and which arise from mutations when carrying out the method. Said method comprises the steps of simultaneously synthesizing a first mixture of equally long oligonucleotides which during synthesis are made ambiguous at the triplets coding for the ammo acid residues at positions 7 and 14 of Leu 17-VIP, and which have a few identical bases at the 3' -end, simultaneously synthesizing a second mixture of equally long oligonucleotides which during synthesis are made ambiguou≤ at the triplet coding for the amino acid residue at position 20 of Leu -VIP, and which have a few identical bases at the 3'-end, these being complementary to .the few bases at the 3 ' -end of the oligonucleotides in the first mixture, mixing said first mixture with said second mixture, whereby the few bases at the 3 '-end of the oligonucleo- tides of the first mixture anneal to the few bases at the 3 ' -end of the oligonucleotides of the second mixture, resulting in a mixture of partially double stranded equally long DNA sequences, which thereafter are enzymatically converted into fully double stranded DNA sequences, subjecting said mixture of double stranded DNA sequences, which include at the 3 ' -end a site for cleavage with a first enzyme and at the 5 '-end a site for cleavage with a second enzyme, to cleavage, simul- taneously or consecutively, with the first and the second enzyme to produce DNA sequences with 3'- and 5 '-ends which are ligatable to the 5'- and 3 ' -ends of similarly cleaved vectors, inserting the thus cleaved DNA sequences into the thus cleaved vectors by ligation to produce a mixture of vectors, which are then transformed into hosts in per se known manner, propagating said hosts to form colonies, which are analyzed one by one to establish those DNA sequences which code for a single protein, propagating separately under expression conditions those hosts which contain vectors having identified protein coding sequences, the C-terminal portions of which correspond to an individual Leu 17-VIP-W29 analog, whereupon the separately expressed proteins are cleaved to release the C-terminal Leu 17-VIP-W29 analogs forming a group of said individual equally long peptides. In one embodiment of this aspect of the inven- tion, wherein W represents Gly, the Leu 17-VIP-W29 analogs which arise from mutations when carrying out the method are Asp 1 -Ala 7 -Ile 14 -Thr 20 -VIP-Gly 29 Tyr 1 -Pro 7 -Thr 14 -Lys 20 -VIP-Gly 29 7 7 8 14 7 C 7 ~ ) Tyr -Thr -Tyr -Thr -Thr -VIP-Gly 3 7 14 20 29 Gly -Thr -Thr -Arg -VIP-Gly z 4 7 14 20 29 Pro -Thr -Arg -Thr -VIP-Gly ~ 7 14 7 0 TO Phe -Thr -Thr -Lys -VIP-Gly 7 R 14 7 f) 9Q Thr -Asn -Arg -Arg -VIP-Gly Pro 7 -Gly 8 -Thr 14 -Lys 20 -VIP-Gly 29 7 8 14 19 20 29 Pro -Gin -lie 1 -Phe 1 -Arg -VIP-Gly^ y Ser 7 -Pro 8 -Ile 14 -Arg 20 -VIP-Gly 29 Ala 7 -Pro 11 -Lys 14 -Lys 20 -VIP-Gly 29 Pro 7 -Pro 12 -Thr 14 -Thr 20 -VIP-Gly 29 Thr 7 -Lys 14 -Asn 15 -Ile 20 -VIP-Gly 29 7 14 1 ζ 70 Pro -He Arg -Lys -VIP-Gly 7 14 20 29 Ala -Lys -Gin 1 -Arg -VIP-Gly zy Ala 7 -Ile 14 -Thr 15 -Lys 20 -VIP-Gly 29 m , 7 _ 14 „. 16 τ 20 „ τυ ,-,, 29 Thr -Lys -His -Lys -VIP-Gly Pro 7 -Ile 14 -His 16 -Thr 20 -VIP-Gly 29 7 14 19 20 29 Thr -He 1 -Phe 1 -lie -VIP-Gly κy 7 14 20 24 29 Pro -Thr -Thr -Lys -VIP-Gly Ser 7 -Lys 14 -Thr 20 -Ile 24 -VIP-Gly 29 7 14 20 28 29 Thr -Thr -Lys -Asp -VIP-Gly 7 14 20 29 Gly -Lys -Lys^ -VIP-Gly , and 7 14 20 29 Ala -Asn -Arg -VIP-Gly . In another embodiment of this aspect of the inven- tion, wherein W represents Gly-Lys-Arg, the Leu 17 -VIP-W 29 analogs which arise from mutations when carrying out the method are π , 2 „ 7 _, 14 _ 20 T7T T , _ 1 29 τ 30 . 3 1 Phe -Pro -Thr -Arg -VIP-Gly -Lys -Arg ττ , 3 m , 7 _ 14 --., 20 -,--.--, _ 1 29 τ 30 _ 31 Val -Thr -Lys -H e -VIP-Gly -Lys -Arg 4 7 1 4 20 29 30 31 Pro -Thr -Thr 1 -Lys -VIP-Gly -Lys° -Arg 5 7 1 4 20 29 30 3 1 Phe -Pro -Thr 1 -Il e u -VIP-Gly -Lys J -Arg J 1 Thr 6 -Ala 7 -Thr 1 4 -Thr 2 0 -VIP-Gly 2 9 -Lys 3 0 -Arg 3 1 Thr 7 -Val 8 -Thr 1 4 -Thr 2 0 -VIP-Gly 2 9 -Lys 3 0 -Arg 3 1 „ 7 π 11 m , 14 _ 20 ττ __. -. , 29 30 _ • 3 1 ■Pro -Pro -Thr -Lys -VIP-Gly -Lys -Arg _. 7 ^ 11 τ l 14 . 20 T7Tr , - 29 _ 30 . 3 1 Pro -Pro -He -Arg -VIP-Gly -Lys -Arg m , 7 _ 13 _ 14 _ 20 _.,___. _ _. 29 _ 30 _ 31 Thr -Arg -Lys -Arg -VIP-Gly -Lys -Arg -Pro 7 -Gln 13 -Lys 14 -Lys 20 -VIP-Gly 29 -Lys 30 -Arg 31 Pro 7 -Pro 13 -Thr 14 -Thr 2 °-VIP-Gly 29 -L s 3 °-Arg 3 X _Pro7-P_,ro13- m Th. r14-A.rg20-_V_IP_-G l ly29-L_ys30-A_rg31 S e er7-A.rg13-T,hr14-Gin15- ^Thr20- T V J ._I_P-G„,ly29-L_ys30-A.rg31 P „ro7-L_ys14-G_ n in15-A_rg20- τ V_I_P_-G_,ly29-L_ys30-A_.rg31 S c er7-L τ ys14-A_sn15-A-,rg20- T V 7 I Tn P-G_ η ly29-L_ys30-A_rg31 m Thr7-L-_ys14-A_sp19-L_ys20- τ V τ I__P- „G 1 ly29-L-ys30-A.rg31 m Th, r7-L τ ys14-G_.ly19-_Th, r20--V.-.-I-..P-,-G__l.y29-L_ys30-A-rg31 Thr 7-Thr14-Ile2 z 0-Asp24-VIP-Gly29-Lys30-Arg3°1 A Λl la7-H-.,e14-A_rg20-A-.sp24--V_I_P-G_,ly29-_Lys30-A Λ rg31 S -er ~ -H T-e 14 -mThr 20 - T Lys24- T V J ._I_P-G -ly29-L_ys30-A_.rg31 _P,ro7- τ H l e14-_Th. r20-T_yr25-_V-.I__P.-G_,ly29-L τ ys30-A Λ rg31 and, S „er7- m Th, r14-H__.e20- τ V r a,l26- T V T _IP_-G_,ly29-L_ys30-A-,rg31 Examples of hosts to be used in the above method are bacteria, e.g. of genus Bacillus or Escherichia coli, and yeasts, e.g. Saccharomyces cerevisiae. Examples of vectors to be used in the above method are bacteriophages and plasmids. In a preferred embodiment of this aspect of the invention, the vectors are plasmids and the hosts are E. coli. As a result of carrying out said preferred embodi¬ ment of the method of the invention there was produced a large number of C-terminally extended Leu 17-VIP analogs, which form an additional aspect of the in- vention. Thus, in an additional aspect of the invention there is provided a C-terminally extended analog of Leu 17-VIP which is chosen from the group consisting of peptides having the amino acid sequence His-Ser-Asp-Ala-Val-Phe-X-Asp-Asn-Tyr-Thr-Arg-Leu-Y- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 -Lys-Gln-Leu-Ala-Val-Z-Lys-Tyr-Leu-Asn-Ser-Ile-Leu- ~ ~ ' ' 17 18 19 20 21 22 23 24 25 26 27 -Asn-W 28 29 wherein X represents Thr, Ser, Pro or ' Ala, Y represents Thr, Lys, He or Ala Z represents Thr, Lys, He or Arg, and W represents Gly or Gly-Lys-Arg, (which resulted from the method and which were ori- ginally planned to be produced) and analogs of Leu 17-VIP having the same amino acid sequence wherein W represents Gly, except for His in position 1 which is sμbstituted by Asp when X = Ala, Y = He and Z = Thr, except for His in position 1 which is substituted by Tyr when X = Pro, Y = Thr and Z = Lys, except for Ser in position 2 which is substituted by Tyr and Asp in position 8 which is substituted by Tyr when X = Thr, Y = Thr and Z = Thr, except for Asp in position 3 which is substituted by Gly when X = Thr, Y = Thr and Z = Arg, except for Ala in position 4 which is substituted by Pro when X = Thr, Y ~ = Arg and Z = Thr, except for Val in position 5 which is substituted by Phe when X = Thr, Y = Thr and Z = Lys, except for Asp in position 8 which is substituted by Asn when X = Thr, Y = Arg and Z = Arg, except for Asp in position 8 which is substituted by Gly when X = Pro, Y = Thr and X = Lys, except for Asp in position 8 which is substituted by Gin and Val in position 19 which is substituted by Phe when X = Pro, Y = He and Z = Arg, except for Asp in position 8 which is substituted by Pro when X = Ser, Y = He and Z = Arg,. except for Thr in position 11 which is substituted by Pro when X = Ala, Y = Lys and Z = Lys, except for Arg in position 12 which is substituted by Pro when X = Pro, Y = Thr and Z = Thr, except for Lys in. osition 15 which is substituted by Asn when X = Thr, Y = Lys and Z = He, except for Lys in position 15 which is substituted by Arg when X = Pro, Y = He and Z = Lys, except for Lys in position 15 which is substituted by Gin when X = Ala, Y = Lys and Z = Arg, except for Lys in position 15 which is substituted by Thr when X = Ala, Y = He and Z = Lys, except for Gin in position 16 which is substituted by His when X = Thr, Y = Lys and Z = Lys, except for Gin in position 16 which is substituted by His when X = Pro, Y = He and Z = Thr, except for Val in position 19 which is substituted by Phe when X = Thr, Y = He and Z = He, except for Asn in position 24 which is substituted by Lys when X = Pro, Y = Thr and Z = Thr, except for Asn in position 24 which is substituted by He when X = Ser, Y = Lys and Z = Thr, except for Asn in position 28 which is substituted by Asp when X = Thr, Y = Arg and Z = Lys, and X = Gly when Y = Lys and Z = Lys, and X = Ala when Y = Asn and Z = Arg, [these analogs can be named with regard to VIP as follows: 1 7 14 20 29 Asp -Ala -lie -Thr -VIP-Gly y Tyr 1 -Pro 7 -Thr 14 -Lys 20 -VIP-Gly 29 7 7 R 14 ?Ω 9 Q Tyr -Thr -Tyr -Thr -Thr -VIP-Gly 3 7 14 20 29 Gly -Thr -Thr -Arg -VIP-Gly 4 7 14 ?Ω 9 Q Pro -Thr -Arg -Thr -VIP-Gly 5 7 14 20 29 Phe -Thr -Thr -Lys -VIP-Gly 7 ft 14 2D 9 Thr -Asn -Arg -Arg -VIP-Gly • Pro 7 -Gly 8 -Thr 14 -Lys 20 -VIP-Gly 29 Pro 7 -Gln 8 -He 14 -Phe 19 -Arg 20 -VIP-Gly 29 Ser 7 -Pro 8 -He 14 -Arg 20 -VIP-Gly 29 7 11 14 20 29 Ala -Pro -Lys -Lys -VIP-Gly 7 1 14 20 29 Pro -Pro -Thr -Thr -VIP-Gl Thr 7 -Lys 14 -Asn 15 -He 20 -VIP-Gly 29 π 7 τ1 14 Λ 15 _ 20 „__ __. 29 Pro -He Arg -Lys -VIP-Gly Ala 7 -Lys 14 -Gln 15 -Arg 20 -VIP-Gly 29 Ala 7 -He 14 -Thr 15 -Lys 20 -VIP-Gly 29 m , 7 _ 14 „. 16 _ 20 T7TT - __, 29 Thr -Lys -His -Lys -VIP-Gly _. 7 τl 14 „. 16 m 20 T7T _, -.-, 29 Pro -He -His -Thr -VIP-Gly 7 14 19 20 29 Thr -lie -Phe -Ile^ -V P-Gly^ Pro 7 -Thr 14 -Thr 20 -Lys 24 -VIP-Gly 29 7 14 20 24 29 Ser -Lys 1 -Thr -lie -VIP-Gly Thr 7 -Thr 14 -Lys 20 -Asp 28 -VIP-Gly 29 7 14 20 29 Gly -Lys -Lys -VIP-Gly and Ala 7 -Asn 14 -Arg 20 -VIP-Gly 29 ] (which arose as mutations when carrying out one run of the preferred embodiment of the method of the in¬ vention) 17 and analogs of Leu -VIP having the same amino acid sequence wherein W represents -Gly-Lys-Arg except for Ser in position 2 which is substituted by Phe when X = Pro, Y = Thr and X = Arg, except for Asp in position 3 which is substituted by Val when X = Thr, Y = Lys and Z = He, except for Ala in position 4 which is substituted by Pro when X = Thr, Y = Thr and Z = Lys, except for Val in position 5 which is substituted by Phe when X = Pro, Y = Thr and Z = He, except for Phe in position 6 which is substituted by Thr when X = Ala, Y = Thr and Z = Thr, except for Asp in position 8 which is substituted by Val when X = Thr, Y = Thr and Z = Thr, except for Thr in position 11 which is substituted by Pro when X = Pro, Y = Thr and Z = Lys, except for Thr in position 11 which is substituted by Pro when X = Pro, Y = He and Z = Arg, except for Leu in position 13 which is substituted by Arg when X = Thr, Y = Lys and Z = Arg, except for Leu in position 13 which is substituted by Gin when X = Pro, Y = Lys and Z = Lys, except for Leu in position 13 which is substituted by Pro when X = Pro, Y = Thr and Z = Thr, except for Leu in position 13 which is substituted by Pro when X = Pro, Y = Thr and Z = Arg, except for Leu in position 13 which is substituted by Arg and Lys in position 15 which is substituted by Gin when X = Ser, Y = Thr and Z = Thr, except for Lys in position 15 which is substituted by Gin when X = Pro, Y = Lys and Z = Arg, except for Lys in position 15 which is substituted by Asn when X = Ser, Y = Lys and Z = Arg, except for Val in position 19 which is substituted by Asp when X = Thr, Y = Lys and Z = Lys, except for Val in position 19 which is substituted by Gly when X = Thr, Y = Lys and Z = Thr, except for Asn in position 24 which is substituted by Asp when X = Thr, Y = Thr and Z = He, except for Asn in position 24 which is substituted by Asp when X = Ala, Y = He and Z = Arg, except for Asn in position 24 which is substituted by Lys when X = Ser, Y = He and Z = Thr, except for Ser in position 25 which is substituted by Tyr when X = Pro, Y = He and Z = Thr, and except for He in position 26 which is substituted by Val when X = Ser, Y = Thr and Z = He, [these analogs can be named with regard to VIP as follows : Phe 2 -Pro 7 -Thr 14 -Arg 20 -VIP-Gly 29 -Lys 30 -Arg 31 Val 3 -Thr 7 -Lys 14 -He 20 -VIP-Gly 29 -Lys 30 -Arg 31 Pro 4 -Thr 7 -Thr 14 -Lys 20 -VIP-Gly 29 -Lys 30 -Arg 31 _,, 5 _. 1 m 14 τl 20 „__ __. 29 _ 30 . 31 Phe. -Pro -Thr -He -VIP-Gly -Lys -Arg Thr 6 -Ala 7 -Thr 14 -Thr 20 -VIP-Gly 29 -Lys 30 -Arg 31 7 8 14 20 29 30 31 Thr -Val -Thr 1 -Thr z -VIP-Gly z -Lys "3 -Arg Pro 7 -Pro i:L -Thr 14 -Lys 20 -VIP-Gly 29 -Lys 30 -Arg 3:L π 7 _. 11 τ1 14 . 20 - 7T --, _.. 29 τ 30 _ 31 Pro -Pro -He -Arg -VIP-Gly -Lys -Arg -. 1 , 13 τ 14 , 20 T7TT - -,, 29 _ 30 _ 31 Thr -Arg -Lys -Arg -VIP-Gly -Lys -Arg - Pro ~ -G - ι in 13 - r Lys 14 -L τ ys 20 - π V τ IP π -G _.ly 29 -L _ys 30 -A _rg 31 _. 7 _ 13 m , 14 _. 20 T7TT - _.. 29 _ 30 _ 31 Pro -Pro -Thr -Thr -VIP-Gly -Lys -Arg 7 13 14 20 29 30 31 Pro -Pro -Thr -Arg -VIP-Gly -Lys -Arg S --er7-A_rg13-_Th, r14-G_.in15- m Thr20- T V 7T I T P--G_,ly29-L_ys30-A_rg31 _Pro7 ^ - τ Lys14-G_,in15-A-,rg20- T V 7X I_P 1 -G--,ly29-L τ ys30-A Λ rg31 7 14 15 90 9Q ^fl ~ ~ Ser -Lys -Asn -Arg -VIP-Gly -Lys -Arg° Thr 7 -Lys 14 -Asp 19 -Lys 20 -VIP-Gly 29 -Lys 30 -Arg 31 Thr 7 -Lys 14 -Gly 19 -Thr 20 -VIP-Gly 29 -Lys 30 -Arg 31 7 14 20 24 29 30 31 Thr -Thr 1 -lie -Asp -VIP-Gly z -Lys J -Arg J A Λ ,la7- τ H l e14-A.rg20-A.sp24- T V 7 I τ P π -G__l.y29- τ Lys30-A_rg31 Ser -H τ ,e14-T_h. r20-L_ys24- ,V JΥ IP n -G_l.y29-L_ys30-A-.rg31 _P.ro7-H÷,e14- m T,hr20-T_yr25- t V rτ I_P-G_l_.y29- τ Lys30-A.rg31 and, S „er7- m T,hr14-H_,e20- T V 7 a,l26- T V 7 I τπ P-G_ 1 ly29- τ Lys30-A_rg31 η j (which arose as mutations when carrying out a second run of the preferred embodiment of the method of the invention) . All the amino acid residues in the C-terminally extended Leu -VIP analogs of the invention are of L-configuration. In a further aspect of the invention there is provided a plasmid containing a gene having a DNA sequence which codes for a C-termmally extended Leu17 -VIP analog of the invention. All the C-terminally extended Leu -VIP analogs of the invention have, besides similar properties to natural human VIP, some useful properties in common, namely, they are ligands at VIP receptors, they are resistant to oxidation and can be kept at acidic pH in solution (due to the replacement of methionine in position 17 of natural VIP" by leucine) , and, since they are C-terminally extended, they exhibit a prolonged effect both in vitro and in vivo, which is considered to be of advantage in view of the extremly short bio¬ logical half-life of VIP. Thus the C-terminally extended Leu -VIP analogs of the invention are useful as potential active ingre¬ dients in new -pharmaceuticals, as well as in cosmetics (for hydration of the skin) . The new pharmaceuticals are intended to be admi¬ nistered i) for the treatment of asthma and of constriction of the upper airways in general, specially for vasodilation in the lungs, 2) as pheripheric vasodilators to be locally applied for i.a. erection of penis, 3) for the treatment of VIPOMA (a VIP producing intestinal tumour which causes death of a human by loss of 5-15 liters of water per day), 4) for the treatment of disorders of blood flow, blood-pressure, intestinal motility and urinary bladder, 5) for potentiation of salivation e.g. for the treat¬ ment of blocked or reduced salivation (e.g. due to administration of antipsychotic drugs or anti- depressants) , and 6) for the stimulation of pancreatic juice secretion. The composition of pharmaceuticals including the active ingredient will depend on the intended route of administration and the specific disorder to be treated. A nasal or oral spray is suitable when asthma is to be treated. Aqueous solutions or tablets for oral administration will be suitable for other indications. In all types of pharmaceuticals the active ingredient is in admixture with pharmaceutically accep¬ table excipients and/or diluents normally used in the field of pharmacy. General strategy to obtain Leu 17-VIP analog-coding DNA sequences cloned in an E. coli expression vector A large number of artificial genes coding for C-termmally extended Leu 17-VIP analogs were planned to be obtained directly as parts of an E. coli plasmid expression vector by performing only one cloning step. The synthetic strategy utilized the principle of a method described originally [Simoncsits, A., Kalman, M. , Cserpan, J. and Kari, C. (1984) Nucleic Acids Res. Sy p. Ser. 1_4_, 321] for obtaining a large number of artificial E. coli promoter derivatives. Briefly, two single-stranded oligodeoxyribonucleotides were chemically synthesized and were annealed [Itakura, K. (1982) TIBS, 1_, 442] through their 3'-terminal comple¬ mentary sequences. The partial duplex obtained so covered the whole Leu 17-VIP coding region and contained additional terminal sequences to be used for cloning. The two oligodeoxyribonucleotides were obtained by ambiguous chemical synthesis so that the mixture of all four activated nucleotide monomers were used at certain predetermined positions (i.e. in triplets coding for the amino acid residues 7, 14 and 20 of Leu 17-VIP) which were outside of the annealing region. The annealed partial duplex was converted by a mutually primed synthesis effected by DNA polymerase (Klenow fragment) into double-stranded DNA mixture containing all possible homoduplexes . These were then cleaved with two different restriction endonucleases the clea¬ vage sites of them being located upstream and downstream of the Leu -VIP coding region and the cleaved mixture was ligated with the appropriately cleaved expression vector pPEX. The ligated mixture was transformed into competent E. coli cells and the recombinants containing Leu 17-VIP related coding sequences were selected by colony hybridization. The positive recombinants were further analyzed by nucleotide sequencing to identify the particular mutations obtained in the cloned Leu 17- -VIP coding region. During the design of the synthetic oligodeoxy¬ ribonucleotides, it was considered that they should code for a methionine preceding the Leu -VIP coding region and for a Gly (VlPa analogs) or a Gly-Lys-Arg ( IPb-analogs) extension at the carboxy terminus. Methionine was included to be able to release the Leu 17-VIP analogs from the fusion protein by CNBr cleavage, while the carboxy terminal extensions could serve as substrate sites for amidation catalysed by peptidylglycine -amidating monooxygenase (PAMase) [Bradburg, A.F., Finnie, M.D.A. and Smyth, D.G. (1982) Nature 298, 686]. The positions of the mixed chemical couplings whithin the nucleotide sequences were chosen to be located in the predetermined X, Y and Z codons so that after performing the steps shown in Scheme 1, the maximum number of amino acid variations be obtained for X, Y and Z. By choosing the first (for codon X) or the second (for codons Y and Z) base of the target codons as mixed positions, four amino acids could be obtained of each three codon variations, yielding altogether 64 possible Leu -VIP analogs after expres¬ sion. The VIPl oligonucleotide, shown as the upper- strand of the partial, annealed duplex in Scheme 1 contains two mixed positions (N), while the lower strand VIP2 oligonucleotide contains only one. The VIP2 oligonucleotide was prepared in two variations carrying information for C-terminal extensions as above (VIP2a and VIP2b oligonucleotides in Scheme 1). To obtain both VlPa and VIPb analogs, two separate experiments were carried out using VIPl + VIP2a or VIPl + VIP2b olignucleotides, respectively. The double stranded DNA mixture obtained by Klenow polymerase reaction contained homoduplex molecules which were separated by cloning into an expression vector. In principle, all recombinants should contain only one type of mutant gene coding for a particular Leu 17-VIP analog. pPEX vector pPEX is a highly efficient E. coli expression vector constructed from a rac fusion [Boros, J., Lukacsovich, T., Baliko, G. and Venetianer, P. (1986) Gene _4_2, 97] promoter vector Lα int 23 named as pPEX by us. The Lα int 23 vector, which expresses under rac promoter control a fusion protein composed of 280 amino acids of E. coli 3-galactosidase and part of the bacterial CAT (chloramphenicol acetyl trans- ferase) was modified by eliminating its unique BamHI site and by replacing its CAT coding region located between Clal and EcoRI sites with a synthetic poly- cloning region. The relevant region of the pPEX vector obtained so is shown in Scheme 2. The vector contains unique BamHI, Kpnl, Sad, Apal and EcoRI sites in the polycloning region and 3-galactosidase gene fusions can be performed using the BamHI and any of the other unique sites. When the Leu 17-VIP coding genes are cloned into the BamHI and EcoRI sites of the pPEX vector, the resulting gene fusions code for 314 and 316 amino acids long β-galactosidase-VIPa and 3-galacto- sidase-VIPb fusion proteins, respectively, of which proteins 284 amino acids are derived from the fusion partner (Scheme 1). EXAMPLE ___ϊ§B___ϊ____=i°5_2__._--k§_ ---^ Leu I _an logs MATERIALS Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs. T4 polynucleotide kinase and Klenow polymerase were from Boehringer, [γ- - 32P]ATP O5000 Ci/mmol) and [α- 32 P]dATP (800 Ci/m ol) were from Amersham, Rabbit antisera directed against BSA-VIP conjugates was provided by Dr. Per Askelof (SBL, Stock¬ holm, Sweden) . Immunoblot assay kit containing anti- -rabbit IgG-alkaline phosphatase conjugate and color development reagents BCIP/NBT was purchased from Bio-Rad. T, RNase was purchased from Calbiochem. 21Ϊ22__i§25 ϊil_>2B-__^ _=2._:i _ CCGGATCCATATGCACTCTGACGCTGTTTTCNCTGACAACTACACT- CGTCTGANAAAACAGCTGGCT (VIPl oligonucleotide), AAGAATTCAGCCGTTCAGGATAGAGTTCAGGTACTTTNTAACAGCC- AGCTGT (VIP2a oligonucleotide), AAGAATTCAACGTTTGCCGTTCAGGATAGAGTTCAGGTACTTTNTA- ACAGCCAGCTGT (VIP2b oligonucleotide) and CAGGGTGAAACGCAGGTCGCCAGCGGC (lac Z 27-mer primer) oligonucleotides were prepared on an automatic DNA synthesizer by the phosphora idite chemistry (Pharmacia Gene Assembler) and were purified by electrophoresis on polyacrylamide gels containing 8 M urea. Oligonucleo- tides were 5 '-phosphorylated with [γ- 32P]ATP by using T4 polynucleotide kinase [Maniates, T. , Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A labora- tory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y] when they were to be used as either hybrid¬ ization probe or sequencing primer. 100 pmol of VIPl oligonucleotide was mixed with 100 pmole of either VIP2a or VIP2b oligonucleotide in 50 μl of water, the mixture was kept at 90°C for 3 min and was let to cool to room temperature in approx. .1 hr period. The solution was made up to 100 μl con¬ taining 10 mM Tris-HCl, pH 8.2, 5 inM MgCl 2 , 50 μM each of dCTP, dGTP and TTP, 5 μl of [α- 32 P]dATP (approx, 62.5 pmol) and 1 μl of 5U/μl Klenow polymerase. After 15 min 10 μl of 1 mM dNTP mixture (containing all four deoxynucleoside 5 '-triphosphates ) was added and the solution was incubated at room temperature for 15 min. The DNA was precipitated with the help of 1 μl of 10 μg/μl yeast carrier tRNA (ctRNA) by ethanol precipitation and purified by 10% acrylamide-8 M urea gel electrophoresis at 400V for 2 hrs (gel thickness 0.4 mm). The gel was radioautographed, the major radio¬ active band was cut out and soaked in 300 μl of 50 mM NaCl - 0.1% SDS solution at 37°C for 12 hrs. The super- natant was phenol extracted and the precipitation was carried out by adding 1 μl of 10 μg/μl ctRNA, 30 μl of 3 M NaOAc, pH 5.2 and 900 μl of ethanol in a liquid nitrogen bath. Centrifugation (14 000 rpm, 3 min) resulted in a radioactive pellet which was washed with ethanol, dried and dissolved in sterile water. 20 pmol of the radioactive Leu 17-VIP-DNA obtained as above was treated with 200 units of BamHI and EcoRI each in 200 μl of 100 mM NaCl, 50 mM Tris HCl, pH 7.5, 10 mM MgCl , 1 mM DTT (high-salt buffer) at 37°C for 20 hrs. The reaction mixture was heated to 60°C for 10 min, phenol extracted (two times), and the DNA was recovered from the waterphase by ethanol pre¬ cipitation as above (two times) followed by ethanol washing, drying and redissolving in sterile water. Analysis of the mixture by 10% acrylamide-8 M urea electrophoresis showed that only approx. 50% of the duplex was cleaved and the fraction of the DNA which had been cleaved by both enzymes was only approx. 30%. Nevertheless, the mixture was used as it was for cloning into BamHI-EcoRI cleaved pPEX vector. 1§^Y§2 _2-- . _EE§$_Z- 2___ £_-- . i___h_Ba^I_and_EcoRI 2 μg of pPEX 13 was treated with 10-10 units of BamHI and EcoRI in 20 μl of high salt buffer at 37°C for 4 hrs. The reaction mixture was applied onto an agarose gel (0.5%) and the electrophoresis was performed in TAE buffer (40 mM Tris-acetate, 2 mM EDTA pH 7.5) at 60V for 1 hr in the presence of ethi- dium bromide. The linear vector band was cut out, electroeluted, phenol extracted twice, ethanol preci- pitated, washed with ethanol and dried. The pellet was dissolved in sterile water. Q 5i22_2 __-_=__ . zYI___lDNA_into_£PEX_vector 0.2 μg BamHI-EcoRI cleaved pPEX vector and approx. 5 pmol BamHI-EcoRI Leu 17-VIP-DNA obtained as above were reacted in 20 μl reaction mixture containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP and 80 units of T4 DNA ligase at 15°C for 12 hrs. The reaction mixture was transformed into JM101 E. coli cells [genotype (supE, thi, Δ(lac-proAB) , [F', traD36, proAB, lad q ZΔM15), Messing, J. , Crea, R. and Seeburg, P.H. (1981) Nucleic Acids Res. _9, 309-321] using a frozen stock [Hanahan, D. in DNA cloning, Vol I., edited by Glover, D.M. IRC Press Limited (1985), pp 109-135, protocol 3]. Ampicillin resistant colonies were picked for colony hybridization [Grunstein, M. and Hogness, D.S. (1975) Proc. Natl. Acad. Sci. USA 72, 3961-3965] with 32 P-labeled VIPl oligonucleotide probe. Approx. 80-90% of the colonies were positive in this test for both VlPa and VIPb mutants. 120 colo- nies of both series were taken for plasmid preparation and sequencing. Plasmid_greparation_and_seguencing A single colony was inoculated into 3 ml of LB- -medium containing 100 μg/ml ampicillin and the culture was shaken for 12-16 hrs at 37°C. Plasmid DNA was prepared by a rapid alkaline extraction procedure [Birnboim, H.C. and Doly, J. (1979) Nucleic Acids Res., 1_, 1513-1523]. The plasmid DNA was further treated with 5 units of T, RNase in 100 μl solution containing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA at 37°C for 30 min followed by phenol extraction and ethanol precipitation. 5 The pellet was taken up in 30 μl of sterile water. Dideoxynucleotide sequencing was performed on Hindlll linearized plasmid template (0.2-0.4 μg) using 5'- 32P-phosphorylated lac Z 27-mer sequencing primer (0.25 pmol) and heat denaturation as described 10 [Hong, G.F., (1982) Bioscience Reports 2 , 907]. Sequencing reactions for 24 clones were performed at the same time. E. coli JM101 cells transformed with pPEX Leu -VIP 15 analog plasmids were grown at 37°C, shaking 250 rpm, in 1 ml LB medium containing 0.1 mg/ml ampicillin. When the cell density reached 0.5 at A=600 n the cells were induced either by addition of IPTG (final concentration 2.5 mM) and shaken at 250 rpm for 4 hours 20 at 37°C or by addition of lactose (final concentration 1%) and shaken at 250 rpm for 20 hours. After induction the cells were collected by centrifugation at 12 000 rpm in an Eppendorf centrifuge for 1 minute, lysed at 100°C for 3 minutes in 300 μl of lysis buffer (0.125 M 25 Tris-HCl pH 6.8, 30% glycerol, 2% SDS, 6 M urea and 1 M 2-mercaptoethanol ) • and 30 μl of non-induced extracts and 30 μl of induced extracts were run on SDS-10% polyacrylamide gels according to Laemmli, U.K. (1970) Nature, 227, 680-685 to monitor the degree of protein 30 expression as shown by coomassie brilliant blue staining of the gels. As estimated from the gels the amount of expressed fusion protein was more than 60% of total cell proteins. 6 μl aliqout of non-induced and 6 μl of 50x diluted induced samples were run on SDS-10% polyacrylamide gels in parallel and half of the gels were stained with coomassie brilliant blue and the other half of __. the gels were electroblotted onto nitrocellulose papers. The blotted nitrocellulose papers were incubated with a polyclonal rabbit serum containing antibodies directed against BSA-VIP, and the immunoblot was developed by treatments with goat anti rabbit IgG conjugated alkaline phosphatase and- BCIP/NBT color development solution (Bio-Rad immuno assay kit) according to the manufacturer's instructions. A very strong immuno- staining was obtained with the fusion proteins con¬ taining Leu -VIP analogs. l__- i___§£_--i2Sl2_Eli^__:_i;i2_?:ti22_ f_25E--_§§§ _!_ l2 l___2_r2--ii-!_-*--..r_~ 17 -Leu zYI____§2_|l2 _I_i_§i 2:_E£2£§i]-_*-___ E. coli JM101 cells carrying the pPEX-Leu -VIP analog plasmids were shaken (250 rpm) in 1 liter LB medium containing 0.1 mg/ml ampicillin at 37°C. When the absorbance at 600 nm reached 0.5 the cells were induced with lactose- (1% final concentration) and shaken at 250 rpm at 37°C for 20 hours. After induction the cells were harvested by centrifugation at 2000 x g, 4°C for 10 minutes, washed with 40 ml of TBS, pH 7.6, and resuspended in 30 ml TBS containing 10 μM PMSF and 0.5 mM EDTA. The cell suspension was then freeze-thawed and sonicated (five 1 minute bursts) in a Branson sonifier, then centrifuged at 17000 x g, 4°C, for 30 minutes. The pellet containing the fusion protein was homogenized in 50 ml 6 M guanidium chloride containing 1% 2-mercaptoethanol according to Goeddel, D.V. et. al. (1979) PNAS (USA), 7_6 / 106-110 and centri- fuged at 60 000 x g for 60 minutes. The clear, amber supernatant containing the fusion protein was diluted to 100 ml with 6 M guanidium chloride - 1% 2-mercapto¬ ethanol and 172 ml TBS was added dropwise with vigorous stirring which rendered most of the fusion protein to precipitate while most of the host proteins remained in solution. The slurry was stirred at room temperatur for 30 minutes before centrifugation at 10 000 x g for 30 minutes at 10°C. The pellet so obtained contains 17 most of the 3-galactosιdase-Leu -VIP analog fusion protein. CNBr_cleavage_of_fusion_protein 17 The partially purified -β-galactosidase - Leu -VIP analog fusion protein was dissolved by homogenization in 25 ml 70% HCOOH and 1.5 g CNBr was added per gram precipitate. The solution was stirred at room tempe- rature for 70 hours and then evaporated at room tem¬ perature under reduced pressure using a Rotavapor. 20 ml 50 mM HCl in methanol was added per gram fusion protein to the residue and stirred at room temperature over night. Then the slurry was centrifuged at 2000 x g, 4°C, for 10 minutes and the supernatant collected. 17 Purifιcation_of_Leu zY_________..__-§logs 17 The supernatant containing the Leu -VIP analog was either 1 ) 17 freeze-dried and the Leu -VIP analog-containing residue was dissolved in 1% TFA and purified on HPLC (C, „-Bondapack, reverse phase) or 2) subjected to further purification as follows before the HPLC step. 17 To the acidic methanol phase containing the Leu -VIP analog solid NaCl (0.2 g/ml ) and 4 volumes of ice cold diethylether were added. 17 The precipitate (containing Leu -VIP analog) was collected on a sintered glass funnel (pore size no 3 ) and dissolved by washing with 5 ml 1 M acetic 17 acid. The dissolved Leu -VIP analog was desalted with a Sephadex G25 column (7 cm long, 2 cm diameter) and freeze-dried before dissolution in 1% TFA and purified on HPLC with a 20%-40% acetonitrile linear 17 gradient in 1% TFA. The Leu -VIP analog was eluted 17 at 33% acetonitrile. The purified Leu -VIP analog was then tested in biochemical and biological assay systems . EXAMPLE: VlPa (X=Thr, Y=Arg, Z=Lys, W=Gly) The fusion protein 3-galactosidase - VlPa was expressed in 1 liter LB medium containing 0.1 mg/ml ampicillin and purified as described above. The fusion protein was cleaved with CNBr and VlPa so obtained was further purified as described above and tested in the following assay systems: 1) Radioimmunoassay (RIA) 2) Radioreceptor assay 3) Stimulation of 3', 5 '-cyclic AMP production 4) Bicarbonate secretion in cat in vivo ) _!_.-_L^i i5!5_i!__:2§§§§Y_i5ϊ^! In the VIP - RIA kit purchased from Penninsula Laboratories (USA) recombinant VlPa is recognized as VIP when measured according to the manufacturer' s recommendation, based on the method of Fahrenkrug, J. and Schaffalitzky De Muckadell, 0. (1977) J. Lab. Clin. Med. 8 . , 1379-1388. 2 ) ___-§____i2:_-.22§E_r2£_.__-__i---§Y Radioreceptor assay was carried out on membranes from rat cerebral cortex using chloramin-T iodinated 125 I-VIP (Hallden, G. et. al. (1986) Reg. Pep. 16, 183-188) at 0.5 nM concentration, studying the displa¬ cement of the labeled VIP by purified VIP from porcine intestine and by recombinant VlPa from E. coli as described by Abens, J. et al. (1984) Peptides _5, 375-377. The affinity of recombinant VlPa was within the experimental error identical to that of VIP purified from porcine intestine with a Kd value of 1.5-1.8 nM at equilibrium, proving that recombinant VlPa is a high affinity ligand at VIP receptors, binding in a manner indistinguishable from that of VIP obtained from other sources. 3 ) §timulation_of_3^ i 5J_-cγclic_AMP_synthesis • Stimulation of 3 ' ,5 '-cyclic AMP ( cAMP) synthesis in tissue slices from rat cerebral cortex by VIP puri¬ fied from porcine and recombinant VlPa was studied to establish whether or not recombinant VlPa, a ligand of the VIP receptor according to the result in 2 ) , behaves as an agonist or an antagonist. The experiments were carried out as follows: Rat cerebral cortical slices (0.4 mm x 0.4 mm) were preincubated in Krebs Ringers bicarbonate buffer, bubbled with 0 2 /C0 2 (95%/5%) (V/V) for 60 minutes at 36°C in the presence of 10 mM theophylline (a phospho- diesterase inhibitor). The tissue slices were then incubated in 600 μl total volume with porcine VIP at 10, 50, 100 nM, 1 and 10 μM concentration for 10 minutes and with recombinant VlPa at 10, 50, 100 and 300 nM concentration (estimated by 1)) for 10 minutes. The incubations were terminated by addition of 150 μl EGTA (100 mM) solution followed by placing the test tubes into a boiling water bath for 3 minutes. The cAMP content was measured according to Brown, B.L. et al. (1972) Adv. Cycl . Nucl. Res. 2 , 25-40. The results indicate that at 10 minutes incubation time recombinant VlPa produced an increase in cAMP levels and thus act as agonist at the VIP-receptor coupled adenylate cyclase, which is thought to mediate the physiological actions of VIP (cf. Rostene, W.H. (1984) Progr. Neurobiol. 2_2, 103-129). 4) Biological_activitγ_of_VIPa The biological activity of recombinant VlPa was also examined in the in vivo assay on stimulation of bicarbonate secretion from cat pancreas according to Mutt, V. and Sδderberg, U. (1959) Arkiv Kern. 15, 63-68. Recombinant VlPa (16 μg injected) was in this assay approximately 70% as efficient as porcine VIP. Recombinant VIPb was, when tested at the same concentration in this assay, at least as efficient as porcine VIP. SCHEME 1 VIPl oligonucleotide Cra^TCCATATGCACTCTGACGCTGTTTTC^TGACA TGTOMπ_yCAATNf C^TGGACTTGAGA CO BamHI X 03»\TCC-ATATG[_ACTCTGACGCTGTTTT[H:TGACAACT rπ GGCCTAGCTATATCTGN^TGOy^^ =1 VlPa and VIPb duplexes 1, BamHI and EcoRI cleavage 2, Cloning into BamHI and E 3, Selection of individual sequencing cont. For all 64 variations: VlPa analogs when W = Gly VIPb analogs when W = Gly-Lys-Arg Scheme 2 Sequence of the polycloninq region of the pPEX vector ω 280 284 a 3-galacto- IleAspSerTrpIleLeu... in sidase ATCGATTCTTGGATCCTCTGATGGTACCTCCTCTGAGCTCTCTGATGGGCCCGAGTATGCGACAGCTGGAATTC- Clal BamHI Kpnl Sacl Apal EcoRI ~ ■ -1 [13 ill ... ι

Claims: CLAIMS 1. A method of simultaneously producing a group of individual C-terminally extended analogs of Leu -VIP (vasoactive intestinal polypeptide) , said group consisting of equally long peptides having the amino acid sequence His-Ser-Asp-Ala-Val-Phe-X-Asp-Asn-Tyr-Thr-Arg-Leu-Y- 1 2 3 4 5 . 6 7 8 9 10 11 12 13 14 -Lys-Gln-Leu-Ala-Val-Z-Lys-Tyr-Leu-Asn-Ser-Ile-Leu- 15 16 17 18 19 20 21 22 23 24 25 26 27 -Asn- 28 29 wherein X represents Thr, Ser, Pro or Ala, Y represents Thr, Lys, lie or Ala Z represents Thr, Lys, lie or Arg, and represents Gly or Gly-Lys-Arg, and of equally long peptides which are also Leu -VIP- - 29 analogs and which arise from mutations when carrying out the method, c h a r a c t e r i s e d by the steps of simultaneously synthesizing a first mixture of equally long oligonucleotides which during synthesis are made ambiguous at the triplets coding for the ammo acid residues at positions 7 and 14 of Leu 17-VIP, and which have a few identical bases at the 3 '-end, simultaneously synthesizing a second mixture of equally long oligonucleotides which during synthesis are made ambiguous at the triplet coding for the amino acid residue at position 20 of Leu -VIP, and which have a few identical bases at the 3 '-end, these being complementary to the few bases at the 3 '-end of the oligonucleotides in the first mixture, mixing said first mixture with said second mixture, whereby the few bases at the 3 '-end of the oligonucleo¬ tides of the first mixture anneal to the few bases at the 3 '-end of the oligonucleotides of the second mixture, resulting in a mixture of partially double stranded equally long DNA sequences, which thereafter are enzymatically converted into fully double stranded DNA sequences, subjecting said mixture of double stranded DNA sequences, which include at the 3 ' -end a site for cleavage with a first enzyme and at the 5 '-end a site for cleavage with a second enzyme, to cleavage, simul¬ taneously or consecutively, with the first and the second enzyme to produce DNA sequences with 3'- and 5 '-ends which are ligatable to the 5'- and 3 '-ends of similarly cleaved vectors, inserting the thus cleaved DNA sequences into the thus cleaved vectors by ligation to produce a mixture of vectors, which are then transformed into hosts in per se known manner, propagating said hosts to form colonies, which are analyzed one by one to establish those DNA sequences which code for a single protein, propagating separately under expression conditions those hosts which contain vectors having identified protein coding sequences, the C-terminal portions of which correspond to an individual Leu 17-VIP-W29 analog, whereupon the separately expressed proteins are cleaved to release the C-terminal Leu -VIP-W analogs forming a group of said individual equally long peptides. 2. A method according to claim 1, wherein W repre- sents Gly and the Leu 17-VIP-W29 analogs which arise from mutations when carrying out the method are Asp 1 -Ala 7 -Ile 14 -Thr 20 -VIP-Gly 29 1 7 14 70 29 Tyr -Pro -Thr -Lys -VIP-Gl 2 7 8 14 20 29 Tyr -Thr -Tyr -Thr -Thr -VIP-Gly 3 7 14 20 29 Gly -Thr -Thr -Arg -VIP-Gly 4 7 14 20 29 Pro -Thr -Arg -Thr -VIP-Gly 5 7 1 20 29 Phe -Thr -Thr -Lys u -VIP-Gly 7 8 14 70 99 Thr -Asn -Arg -Arg -VIP-Gl 7 8 14 20 29 Pro -Gly -Thr -Lys -VIP-Gly 7 8 14 19 20 29 Pro -Gin -lie 1 -Phe -Arg^ -VIP-Gl S ,,er7-P_ro8-l_,ie14-A.rg20--V._.I_P_-G-,,ly29 A _,la7-P_ro11-L_ys14-L_ys20- T V_I__P-G_ 1 ly29 Pro 7 -Pro 12 -Thr 14 -Thr 20 -VIP-Gly 29 m Th. r7-L_ys14-A..sn15-_l.ie20-_V_I__P-G_.ly29 Pro 7 -Ile 14 Arg 15 -Lys 20 -VIP-Gly 29 A -.,la7-L--ys14-G_ π in15-A_rg20-„VI--.P--,-G_l,y29 Ala 7 -Ile 14 -Thr 15 -Lys 20 -VIP-Gly 29 Thr 7 -Lys 14 -His 16 -Lys 20 -VIP-Gly 29 Pro 7 -Ile 14 -His 16 -Thr 20 -VIP-Gly 29 7 14 19 20 29 Thr -Ile 1 -Phe 1 -Ile κυ -VIP-Gly zy 7 14 20 24 29 Pro -Thr -Thr -Lys -VIP-Gl Ser 7 -Lys 14 -Thr 20 -Ile 24 -VIP-Gly 29 Thr 7 -Thr 14 -Lys 20 -Asp 28 -VIP-Gly 29 7 14 20 29 Gly -Lys -Lys -VIP-Gly , and 7 14 20 29 Ala -Asn -Arg -VIP-Gly . 3. A method according to claim 1, wherein W repre- sents Gly-Lys-Arg and the Leu 17-VIP-W29 analogs which arise from mutations when carrying out the method are 2 7 14 20 29 ~ , 0 ~ l Phe -Pro -Thr -Arg -VIP-Gly -Lys -Arg Val 3 -Thr 7 -L y s 14 -Ile 20 -VIP-Gly 29 -Lys 30 -Arg 31 Pro 4 -Thr 7 -Thr 14 -Lys 20 -VIP-Gly 29 -Lys 30 -Arg 31 Phe 5 -Pro 7 -Thr 14 -Ile 20 -VIP-Gly 29 -Lys 30 -Arg 31 Thr > 7 14 20 29 30 31 -Ala -Thr -Thr -VIP-Gly z -Lys JU -Arg J1 Thr '-Val 8 -Thr 14 -Thr 20 -VIP-Gly 29 -Lys 30 -Arg 31 Pro '-Pro11- m Th, r14-L_ys20- τ V. τ I_P-G_.ly29-L_ys30-A n rg31 Pro' '-Pro11- τ l,ie14-A Λ rg20-„V_IP_-G_-ly29-L_ys30-A_rg31 ' 13 _ 14 .. 20 „__ __. 29 _ 30 Λ 31 Thr' -Arg -Lys -Arg -VIP-Gly -Lys -Arg To -G -,,in13-L_ys14-L τ ys20--V..I__P-G_,ly29-L_ys30-A A rg31 7 13 14 20 ' 29 30 31 Pro -Pro -Thr 1 -Thr -VIP-Gly -Lys J -Arg _P,ro7-_P.ro13-T_h. r14-A n rg20-„VI__P-G__l.y29-_Lys30-A_rg.31 7 1 ^ 14 1R 9fl 9 ^D Ser -Arg- -Thr -Gin -Thr -VIP-Gly -Lys -Arg 31 _P,ro7-L τ ys14-G_,in15-A_rg20-.V 7T I-P-,-G__l.y29-_Lys30-A_rg31 S c er7-L τ ys14-A,sn15-A Λ rg20-.V.I__P-G_.ly29-_Lys30-A Λ rg31 Thr 7 -Lys 14 -Asp 19 -Lys 20 -VIP-Gly 29 -Lys 30 -Arg 31 Thr 7 -Lys 14 -Gly 19 -Thr 20 -VIP-Gly 29 -Lys 30 -Arg 31 m Th u r7-T_.hr14-_l_i.e20-A Λ sp24-„V τ IP_.-G__l.y29-L_ys30-A_rg31 A -.,la7-l τl ie14-A,.rg20-A,sp24- τ V τ I τ P τ -,-G_.ly29- τ Lys30-A,rg31 S -,er7-l τl ie14-T_.hr20-L τ ys24-„V_I-.„P-G_.ly29-L_ys30-A.rg31 P n ro7-l τ -ie14-T_.hr20-T_yr25-„V_IP_-G_,ly29-_Lys30-A Λ rg31. and, S „er7- m Thr14-_l..i.e20- T V 7 al.26-„VI τ P rι -^G,,ly29-_Lys30-A-.rg31 4 . A method according to any one of claims 1 -3 , c h a r a c t e r i s e d in that the vectors are plasmids and the hosts are E . coli . 5 . A C-termmal ly extended analog of Leu 1 7 -VIP c h a r a c t e r i s e d in that it is chosen from the group consisting of peptides having the amino acid sequence His-Ser-Asp-Ala-Val-Phe-X-Asp-Asn-Tyr-Thr-Arg-Leu-Y- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 -Lys-Gln-Leu-Ala-Val-Z-Lys-Tyr-Leu-Asn-Ser-Ile-Leu- 15 16 17 18 19 20 21 22 23 24 25 26 27 -Asn-W 28 29 wherein X represents Thr, Ser, Pro or Ala, Y represents Thr, Lys, lie or Ala Z represents Thr, Lys, lie or Arg, and W represents Gly or Gly-Lys-Arg, and 1 7 14 20 29 Asp -Ala -lie 1 -Thr -VIP-Gly :? 1 7 1 20 29 Tyr -Pro '-Thr ~ ~ -Lys -VIP-Gly Tyr 2 -Thr 7 -Tyr 8 -Thr 14 -Thr 20 -VIP-Gly 29 Gly 3 -Thr 7 -Thr 14 -Arg 2 °-VIP-Gly 29 Pro 4 -Thr 7 -Arg 14 -Thr 20 -VIP-Gly 29 ~~ 7 14 20 29 Phe -Thr'-Thr 1 -Lys z -VIP-Gly 7 H 14 9(1 99 Thr '-Asn -Arg -Arg z -VIP-Gly ~ ~ -Gl 8 14 20 , 29 Pro'-C y -Thr 1 -Lys -VIP-Gly Pro - Gln 8 -Ile 14 -Phe 19 -Arg 20 -VIP-Gly 29 7 8 14 20 29 Ser -Pro -He 1 -Arg -VIP-Gl 7 11 14 9(1 99 Ala -Pro -Lys -Lys -VIP-Gl 7 12 14 20 29 Pro -Pro -Thr -Thr -VIP-Gl Thr 7-Lys1 1 4-Asn15-Ile20 u -VIP-Gl 29 7 14 15 20 29 Pro -He 1 Arg 1 -Lys -VIP-Gl 7 14 15 20 29 Ala -Lys 1 -Gin 1 -Arg z -VIP-Gly^ 7 14 15 20 29 Ala -lie 1 -Thr 1 -Lys -VIP-Gly 7 14 16 20 29 Thr -Lys -His -Lys -VIP-Gl Pro 7 -Ile 14 -His 16 -Thr 20 -VIP-Gly 29 Thr 7-Ile1 1 4-Phe1 1 9-Ile20 u -VIP-Gly29 y 7 14 20 24 29 Pro -Thr -Thr -Lys -VIP-Gly Ser'-Lys 14-Thr20-lie24-VIP-Gl 29 Thr 7 - Thr 14 -Lys 20 -Asp 28 -VIP-Gly 29 Gly 7 - Lys 14 -Lys 20 -VIP-Gly 29 Ala ' Asn 14 -Arg 20 -VIP-Gly 29 and Phe k - Pro 7-Thr1 1 4-Arg2^0-VIP-Gly z 29-Lys3 J 0-Arg3°1 Val 3 - Thr 7 -L y s 14 -Ile 20 -VIP-Gly 29 -L Y s 30 -Arg 31 4 Pro - Thr 7 -Thr 14 -Lys 20 -VIP-Gly 29 -Lys 30 -Arg 31 Phe 5 - Pro 7 -Thr 14 -Ile 20 -VIP-Gl Y 29 -Lys 30 -Arg 31 Thr 6 - Ala 7 -Thr 14 -Thr 20 -VIP-Gly 29 -Lys 30 -Arg 31 Thr 7 -Val 8 -Thr 14 -Thr 20 -VIP-Gly 29 -Lys 30 -Arg 31 π Pro7-P_ro11-_Th. r14- τ Lys20-.V.I_P_-G_-ly29-L_ys30-A.rg31 _P.ro7-_Pro11-H τl e14-A.rg20-V 7 I XT P--G__l.y29-L_ys30-A.rg31 m Th, r7-A,rg13-L τ ys14-A,rg20--V-.I--.-P-,-G_,_l.y29-L τ ys3 * 0-A,rg31 P _.ro7-G_i_.n13-L_ys14-L_ys20-V„I_P_-G_l η y29-L_ys30-A Λ rg31 _P.ro7-_P.ro13- m Thr14- m T,hr20-„V_IP_-G__l.y29-L_ys30-A_,rg31 7 13 14 20 29 30 31 Pro -Pro -Thr -Arg -VIP-Gly -Lys -Arg Ser 7 -Arg 13 -Thr 14 -Gln 15 -Thr 20 -VIP-Gly 29 -Lys 30 -Arg 31 7 14 15 20 29 30 "31 Pro -Lys -Gin -Arg υ -VIP-Gly -Lys J -Arg J1 Lys 14-Asn1 1 5-Arg2 z 0-VIP-Gly29-Lys3 "3 0-Arg3 J 1 Lys 1 -Asp 19 -Lys 20 -VIP-Gly 29 -Lys 30 -Arg 31 Lys 14 -Gly 19 -Thr 20 -VIP-Gly 29 -Lys 30 -Arg 31 Thr 14 -Ile 20 -Asp 24 -VIP-Gly 29 -Lys 30 -Arg 31 Ile 14 -Arg 20 -Asp 24 -VIP-Gly 29 -Lys 30 -Arg 31 Ile 14 -Thr 20 -L y s 24 -VIP-Gl y 29 -Lys 30 -Arg 31 Pro' Ile 14 -Thr 20 -T y r 25 -VIP-Gl y 29 -L s 30 -Arg 31 and Ser 7 -Thr 14 -Ile 20 -Val 26 -VIP-Gl y 29 -L s 30 -Arg 31 wherein all the amino acid residues are of L-confi- guration. 6. A plasmid containing a gene coding for a C-ter- mmal.llyy eexxtteennddeedd aannaalloogg ooff LLeeuu 1 177--VVIIPP,, cc hh aa rr aa cc - t e r i s e d b y a DNA sequence whi .cchh codes for a CC--tteerrmmimnaall!ly extended analog of Leu 17-VIP as defined in claim 5

Inventors: BARTFAI TAMAS, CSERPAN IMRE, KALMAN MIKLOS, SIMONCSITS ANDRAS, TJOERNHAMMAR MARIE-LOUISE, BARTFAI, TAMAS, CSERPAN, IMRE, KALMAN, MIKLOS, SIMONCSITS, ANDRAS, TJOERNHAMMAR, MARIE-LOUISE
Applicants: TRION FORSKNING & UTVECKLING, TRION FORSKNING- OCH UTVECKLINGS AKTIEBOLAG
Application year: 1988
Main classification: C12N15
IPC codes: A61K38, A61P1, A61P9, A61P11, A61P13, A61P15, A61P25, A61P35, C07H21, C07K1, C07K14, C07K19, C12N15, C12P21, C12R1