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The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) domain of PAM in the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-alpha-hydroxylating monooxygenase (PHM) domain of PAM.Disclosed are: a recombinant C-terminal α-amidated enzyme derivative which lacks the formation of at least one disulfide bond among five disulfide bonds occurring in a C-terminal α-amidated enzyme derived from Xenopus laevis; DNA encoding the derivative; an expression vector carrying the DNA; a bacterium...Disclosed are: a recombinant C-terminal α-amidated enzyme derivative which lacks the formation of at least one disulfide bond among five disulfide bonds occurring in a C-terminal α-amidated enzyme derived from Xenopus laevis; DNA encoding the derivative; an expression vector carrying the DNA; a bacterium Escherichia coli transformed with the expression vector; and a method for producing the derivative by using the bacterium Escherichia coli., wherein said enzyme is AE-I [1-321] (C40A and C85A) (SEQ ID NO: 27), AE-I [1-321] (C252A and C274A) (SEQ ID NO: 33), AE-I [1-321] (C40A and C85A, C252A and C274A) (SEQ ID NO: 37), AE-I [8-321] (C145A) (SEQ ID NO: 25), or AE-I [8-321] (C145A, C40A, and C85A) (SEQ ID NO: 35).coli, and the inclusion body obtained is solubilized under a non-reducing condition and subjected to a refolding procedure.Specifically the above problem may be resolved by the following [1] to [7]: [1] A recombinant C-terminal α-amidating enzyme derivative comprising: (a) a polypeptide having an amino acid sequence in which at least one cysteine residue selected from the group consisting of cysteine residues at positions 6, 145, 40, 85, 252, and 274 has been altered in the amino acid sequence set forth in SEQ ID NO: 2; or (b) a polypeptide having an amino acid sequence in which one or a few amino acid residues out of the amino acid residues other than the cysteine residue have been deleted, substituted, or added in the altered amino acid sequence described in the above (a) and having the activity of C-terminal α-amidating enzyme; wherein at least one disulfide bond has not been formed out of the bonds between the cysteine residues at positions 6 and 145, between the cysteine residues at positions 40 and 85, and between the cysteine residues at positions 252 and 274.

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[5] DNA encoding the recombinant C-terminal α-amidating enzyme derivative according to any one of the above [1] to [4].[8] A method of producing the recombinant C-terminal α-amidating enzyme derivative according to any one of the above [1] to [4], said method comprising the steps of culturing the Escherichia coli according to the above [7], allowing the recombinant C-terminal α-amidating enzyme derivative to be expressed, and then recovering the derivative thus obtained.shows a comparison of the amino acid sequence of the Xenopus laevis C-terminal α-amidating enzyme (SEQ ID NO: 39) and that of a rat C-terminal α-amidating enzyme (SEQ ID NO: 40).Peptidylglycine-alpha-hydroxylating monooxygenase, PHM, and peptidyl-alpha-hydroxyglycine alpha-amidating lyase, PAL, are stitched together in most vertebrates, kinetic studies with purified PHM and PAL revealed a higher affinity of PHM for its peptide substrate and a higher turnover number for PAL the the peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) is located on a domain of peptidylglycine alpha-amidating monooxygenase (PAM).Peptidylglycine-alpha-hydroxylating monooxygenase, PHM, and peptidyl-alpha-hydroxyglycine alpha-amidating lyase, PAL, are stitched together in most vertebrates, kinetic studies with purified PHM and PAL revealed a higher affinity of PHM for its peptide substrate and a higher turnover number for PAL in the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-alpha-hydroxylating monooxygenase (PHM) domain of PAM.

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