KR850700134A - A synthetic polypeptide corresponding to a thermally labile enterotoxin region of Escherichia coli, a composition thereof, and a method for producing the same. - Google Patents

Abstract

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Description

Synthetic polypeptides corresponding to heat-labile enterotoxin domains of Escherichia coli, compositions thereof and methods of manufacture

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Claims (25)

(LTB) of the heat-labile enterotoxin of Escherichia col and a sequence at a position comprising about 35 to about 95 positions from the amino terminus, i.e., the 21 residue signal polypeptide of the B-subunit Corresponding to about 10 to about 35 amino acid residues. The synthetic polypeptide of claim 1, comprising about 15 to about 30 amino acid residues corresponding to a sequence from about 55 to about 85 positions of said B-subunit. The synthetic polypeptide of claim 1, comprising about 20 to about 25 amino acid residues corresponding to a sequence of about 60 to about 85 positions of said B-subunit. (A) an amino acid residue sequence corresponding to at least 14 carboxy-terminal residues of a thermostable enterotoxin of Escherichia coli, and (b) an amino acid residue sequence corresponding to An amino acid residue sequence corresponding to a residue at a position comprising about 35 to about 95 positions from the amino-terminus of the heat-labile enterotoxin B-subunit of Escherichia coli, i.e., the 21 residue signal polypeptide of the B-subunit ≪ / RTI > and comprising about 25 to about 55 amino acid residues. 5. The conjugate polypeptide of claim 4, wherein the two sequences are joined together by peptide bonds formed between amino-terminal residues of heat-stable enterotoxin sequences and heat-labile enterotoxin B-subunit sequences. 6. The conjugate polypeptide according to claim 5, which is expressed from left to right in the direction of the carboxy-terminal at the amino-terminus, corresponding to the following structural formula. MetValIleIleThrpheMet (Lys) SerGlyGlu (Ala) ThrPheGlnValGluValProGlySerGlnHislleAspSerGlnLysAsnThrPheTyrCysCysGluLeuCysCysTyr (Asn) ProAlaCysAlaGlyCysAsn (Tyr) In this formula, the amino acid residues in the parentheses can be replaced with the immediately preceding amino acid residues in the sequence, respectively. Amino-terminal to carboxy-terminal method, from left to right, corresponding to the general formula: (X) _n - (A) _o- (Z) _r -B- (Z) _s- (A) _p- (Y) _m wherein X, Y and Z, if present, Arg residues, or (b) an additional amino acid residue selected from the group consisting of Asp and Glu residues; &Quot; n " and " m " are integers of 0, 1, 2, 3, or 4 where one or both of & If one or both of m is other than 0, there are corresponding X and Y residues wherein the average number of Y and Y residues present per polypeptide is equal to the value of "n" and "m"respectively; r or s is an integer of 0, 1 or 2, and if either or both of r " and " s " are 0, then the corresponding Z is absent and if " r " and &Quot; r " or " s ", provided that the average number of Z moieties per conjugated polypeptide molecule is equal to the value of & And the other is 0, and there is no Z corresponding to " r " or " s "&Quot; o " and " p " are integers of 0 or 1, and there is no corresponding A when one of " o & Can not have; A is an amino acid residue at a position from about 35 to about 95 positions from the amino-terminus of the thermo-labile enterotoxin B-subunit of Escherichia coli, i.e., the 21 residue signal polypeptide of the B-subunit A polypeptide comprising from about 10 to about 35 amino acid residues corresponding to the sequence; B is a polypeptide containing up to 18 amino acid residues corresponding to at least 14 carboxy-terminal residue sequences of thermostable enterotoxin of Escherichia coli. 8. The compound of claim 7 wherein " n " is 2, X is Lys, " o " is 1, " r ", " s ", " p & A is labeled from left to right in terms of the carboxy-terminal orientation from the amino terminus, MetValIleIlePhrPheMet (Lys) SerGlu (Alua) ThrPheGlnValGluValProGlyserGlnHisIleAspSerGlnLys; B is expressed from left to right in the direction of amino-terminal to carboxy-terminal, AsnThrPheTyrCysGluLeuCysCysTyr (Asn) A polypeptide corresponding to ProAlaCysAlaGlyCysAsn (Tyr), wherein the amino acid residues in the parentheses in the structural formulas are each an amino acid residue that can be replaced with the immediately preceding amino acid in the sequence. Corresponds to an amino acid residue sequence at a position comprising about residue 35 to about 95 positions from the amino terminus of the thermo-labile enterotoxin B-subunit of Escherichia coli, i.e., the 21 residue signal peptide of the B-subunit ≪ / RTI > comprising from about 10 to about 35 amino acid residues of at least one polypeptide repeat unit. 10. The polymer according to claim 9, wherein the repeating unit is bonded to each other by a cystine disulfide bond formed by an oxidation reaction of Cys residues existing at both the amino terminal end and the carboxy terminal end of the repeating unit polypeptide before the oxidation reaction. 11. The polymer of claim 10, which is a linear homopolymer. The method of claim 9, further comprising a plurality of second polypeptide repeat units, denoted left to right in the direction of the carboxy-terminal end at the amino-terminus, comprising a polypeptide sequence corresponding to the general formula: The second polypeptide repeating unit is present in a molar ratio of 1:10 to 10: 1 and the first polypeptide repeating unit and the second polypeptide repeating unit are bonded to each other by a cystine disulfide bond formed by oxidation of a Cys residue present in each of these repeating units ≪ / RTI > is a random network copolymer. Wherein the Asn and Tyr amino acid residues in parentheses are residues that can be replaced with respect to the amino acid residues immediately preceding the sequence, respectively; a, b, c, d, e and f and g, h, i, j, k and 1 are each an integer of 0 or 1, provided that any of a to f or g to l is 0 Corresponding or Or or There is no group, and one If the group does not exist, The sulfur atom of the non-cleaved Cys residue forms a cystine disulfide bond, and when any of a to f or g to l is 1, the corresponding or The group is present; Groups are independently of each other the same or different residues bound to the sulfur of the Cys residue and are hydrogen, an alkyl group having from 1 to about 4 carbon atoms or a substituted allyl group having from 2 to about 4 carbon atoms; Are amino acid residues that are the same or different from each other, which can be substituted for the immediately preceding Cys residue shown in the above general formula, and which have one neutral side chain; At least four of a to f and at least four of g to l are zero. The method according to claim 9, further comprising a plurality of second polypeptide repeating units corresponding to the following formula, denoted left to right in carboxy-terminal orientation at the amino-terminus, wherein the first polypeptide repeat unit and the second polypeptide repeat unit are 1:10 To 10: 1, and wherein the first polypeptide repeat unit and the second polypeptide repeat unit are random network polymers wherein the first polypeptide repeat unit and the second polypeptide repeat unit are bonded together by a cysteine disulfide bond formed by an oxidation reaction of a Cys residue present in each of these repeat units . (Tyr) wherein the Asn and Tyr amino acid residues in parentheses are residues that can be replaced with respect to the amino acid residues immediately preceding the sequence, respectively; a, b, c, d, e and f and g, h, i, j, k and 1 are each an integer of 0 or 1, provided that any of a to f or g to l is 0 Corresponding or Or or There is no group, and one If no group exists The sulfur atom of the non-cleaved Cys residue forms a cystine disulfide bond, and when any of a to f or g to l is 1, the corresponding Or However, Independently of one another, denote hydrogen, an alkyl group having from 1 to about 4 carbon atoms or a substituted alkyl group having from 2 to about 4 carbon atoms as the same or different residues bonded to the sulfur atom of the Cys residue, Quot; is an amino acid residue having one side chain which is neutral as the same or different amino acid residues that can be substituted for the immediately preceding Cys residue shown in the above general formula; At least four of a to f and at least four of g to l are zero. 14. The polymer of claim 13, wherein a to f and g to l are zero. 10. The conjugate of claim 9, which is a peptide conjugate peptide conjugated to a second polypeptide, said conjugated polypeptide repeating unit labeled from left to right in the direction from the amino terminus to the carboxy terminus, corresponding to the following formula and containing from about 25 to about 55 amino acid residues, Wherein the complex polypeptide repeating units are linked together by a cysteine disulfide bond formed by an oxidation reaction of a Cys residue present in each repeating unit, wherein the repeating units comprise from about 10 to about 35 amino acid residue polypeptide sequences ≪ / RTI > _{(X) n - (A)} o - (Z) r -B- (Z)% - (A) p - (Y) m Wherein X, Y and Z, when present, are selected from (a) Lys and Arg residues, or (b) an amino acid residue selected from Asp and GIu residues; N " and " m " are integers of 0, 1, 2, 3, or 4 and either or both of & If one or both of " m " is a value other than 0, then corresponding X and Y residues are present, the average number of Y and Y residues present per polypeptide is equal to the corresponding value of " n &;Quot; r " and " s " are integers of 0, 1, or 2 and do not correspond to Z when one or both of r and s are 0, The corresponding number of Z residues per repeating unit of complex polypeptide is equal to the value of "r" or "s", provided that if either one of "r" or "s" The other of " s " is 0, and there is no Z corresponding to " r " or " s " &Quot; o " and " p " are integers of 0 or 1, and there is no corresponding A when one of " o ≪ / RTI > A is the above-mentioned first polypeptide sequence containing about 10 to 35 amino acid residues; B is a second polypeptide from amino-terminus to left-to-right in the orientation of the carboxy terminus and comprising an amino acid sequence corresponding to the general formula: [Wherein, the Asn and Tyr amino acid residues in the parentheses are amino acid residues that can be substituted for the amino acid residues immediately before in the sequence; a, b, c, d, e and f and g, h, i, j, k and 1 are each an integer of 0 or 1, provided that any of a to f or g to l is 0 Corresponding or Or or There is no group, and one If the group does not exist, The sulfur atom of the non-cleaved Cys residue forms a cystine disulfide bond, and when any of a to f or g to l is 1, the corresponding or The group is present; Groups independently of one another are the same or different residues bound to the sulfur atom of the Cys residue; An alkyl group having 1 to 4 carbon atoms or a substituted alkyl group having 2 to 4 carbon atoms; Are amino acid residues that are the same or different from each other, which can be substituted for the immediately preceding Cys residue shown in the above general formula, and which have one neutral side chain; at least four of a to f and at least four of g to l are 0. [ 16. A polymer according to claim 15, wherein said complex polypeptide repeating unit, denoted left to right, at the amino-terminal end in a carboxy termini is selected from the following polypeptide groups. (a) MetValIleIleThrPheMetSerGlyGluThrPheGlnValProGlySerGlnHisIleAspSerGlnLysAsnThrPheTyrCysCysGluLeuCysTyrProAlaCysAlaGlysAsn, (b) LysLysMetValIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysValGluValProGlySerGlnHisIleAspSerGlnLysAsnThrPheTyrCysCysCysGluLeuCysCysTyrProAlaCysla GlyCysn, (c) AsnThrGlnIleTyrThrIleAspLysIleLeuSerTyrThrGluSerMetAlaGlyLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn, (d) Lt; / RTI > (e) MetSerGlyGluThrPheGlnValGluValProGlyAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn, (f) PheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisisIleAspSerGlnLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn, (g) PheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsnThrPheTyrCysCysGluLeu CysCysTyrProAlaCysAlaGlyCysAsn, (H) SerGlnHisIleAspSerGlnLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn, (i) TyrThrGluSerMetAlaGlyLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaGlyCysAsn, (j) AsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsnasnThrGlnIleTyrThrIleAsnAspLysIleLeuSerTyrThrGluSerMetAlaGlys. And a plurality of polypeptide repeating units corresponding to the general formula shown below from the left to the right in the carboxy terminal direction at the amino terminus, and these repeating units are constituted by a polypeptide cystine bond formed between the Cys residues present in the repeating unit Bonded network polymer. (X) _n MetValIleIleThrPheMet (Lsy) SerGlyGlu (Ala) ThrPheGlnValGluValProGlySerGlnHisIleAspSreGlnLysAsnThrPheTyrCysCysGluLeuCysCysTyr (Asn) ProAlaCysAlaGlyCysAsn (Tyr) _m Wherein X and Y, when present, are selected from (a) Lys and Arg residues, or (b) an amino acid residue selected from Asp and GIu residues; N and m are integers of 0, 1, 2, 3, or 4, and when one or both of " n " and " m " are 0, the corresponding X and Y are absent, And " m " or the non-zero, the corresponding X and Y moieties are present and the average number of X and Y present per repeating unit of the polypeptide is equal to the corresponding value of " n &; The amino acid residues in parentheses are residues that can be substituted for the immediately preceding amino acid residues in the sequence of the general formula, respectively. 18. The compound of claim 17 wherein " n " is 2; A network polymer wherein X is Lys and " m " 18. The network polymer of claim 17 wherein " n " and " m " Corresponding to a sequence at a position of about 35 to about 95 positions from the amino-terminus of the heat-labile enterotoxin B-subunit of Escherichia coli, i.e., the 21-residue signal peptide of the toxin, To about 35 amino acid residues wherein said polypeptide is conjugated to a carrier as a repeat unit of a conjugate or polymer and is present in a physiologically acceptable chelating agent as an active ingredient Subunit, and wherein the unit dose is injected into a host mammal to cause the generation of an antibody that immunoreacts with said B-subunit. 21. The inoculum of claim 20, wherein said polypeptide is coupled to a carrier as a conjugate. 21. The inoculum of claim 20, wherein said polypeptide is present as polymer repeat units. 23. The inoculum of claim 22, which is capable of protecting said host from heat-labile enterotoxins of Escherichia coli. Corresponding to a sequence at a position containing about 35 to about 95 positions from the amino terminus of the thermo-labile enterotoxin B-subunit of Escherichia coli, i.e., the 21 residue signal peptide of the toxin, from about 10 to about B-cell of a heat-labile enterotoxin of Escherichia coli induced by injection of an effective amount of a synthetic polypeptide containing 35 amino acid residues and linked to a carrier as a repeating unit of a conjugate or polymer and present in the inoculum, An antibody that immunoreacts with a subunit. 27. The antibody of claim 24, which is expressed from left to right in a carboxy-terminal orientation from the amino-terminus, to a polypeptide corresponding to a sequence selected from the following sequences. (a) MetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaGlyCysAsn, (b) LysLysMetValIleIleThrPheMetSerGlyGluThrPheGlnValGuValProGlySerGlnHisIleAspSerGlnLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaGlyCysAsn, (c) AsnThrGlnIleTyrThrrIleAsnAspLysIleLeuSerTyrThrGluSerMetAlaGlyLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn, (d) GlnIleTyrThrIleAsnAspLysIleLeuSerTrThrGluSerMetAlaGlyLysArgGluMetValIleIleThrPheMetSerGlyGluThrPheGlnValGluValProGlycasnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn, (e) MetSerGlyGluThrPheGlnValProGlyAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn, (f) PheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlysAsn, (g) PheGlnValGluValProGlySerGlnHisIleAspSerGlnLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn, (h) SerGlnHisIleAsSerGlnLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaGlyCysAsn, (i) TyrThrGluSerMetAlaGGlyLysAsnThrPheTyrCysCysGluLeuCysCysTyrProAlaGlyCysAsn, (j) AsnThrPheTyrCysCysGluLeuCysCysTyrProAlaCysAlaglyCysAsnAsnThrGlnIleTyrThrIleAsnAspLysIleLeuSerTyrThrGluSerMetAlaGlyLys, (k) AsnThrGlnIleTyrThrIleAsnAspLysIleLeuSerTyrThrGluSerMetAlaGlyLys, (l) AspLysIleLeuSerTyrThrGluSerMetAlaGlyLysArgGluMetValIleIleThrPhe, (m) TyrThrGluSerMetAlaGlyLysArgGlu, (n) TyrThrGluSerMetAlaGlyLysArgGluMetIleIleThrPhe, (o) GlyLysArgGluMetValIleThrPheMetSerGlyGluThrPheGlnValGlu, (p) IleThrPheMetSerGlyGluThrPheGlnValGluValProGlySerGlSerGln, (q) PheMetSerGlyGluThrPheGlnValGluValProGlySerGlnHisIleAspSerGlnLysLysAlaIleGluArgMetLysAspThrLeu, (r) ValGluValProGlySerGlnHisIleAspSerGlnLysAlaIleGlu, and gt; ※ Note: It is disclosed by the contents of the first application.