Peptidylglycine alpha amidating enzyme

Posted by / 11-Dec-2017 14:01

The glycine-extended model peptide X-Gly (mean = Ala-Ile-Gly-Val-Gly-Ala-Pro) was used as substrate for the purified enzyme.

As a method for resolving this problem, a method of using E. Xenopus laevis C-terminal a-amidating enzyme (peptidyl-glycine alpha-amidating monooxygenase I, EC 1.14.17.3) in large quantities by a recombinant technology in E. However, most of the C-terminal a-amidating enzyme and derivatives thereof expressed by this method are forming inclusion bodies (a mass of inactive protein having the same amino acid sequence but does not have a higher-order structure, and thus is called insoluble granules) in and do not exhibit the activity of the C-terminal a-amidating enzyme.

coli by a method wherein a recombinant C-terminal a-amidating enzyme derivative of which amino acid sequence has been altered so as to prevent the formation of at least one specific disulfide bond of the five disulfide bonds that can be formed during refolding in the production, using a gene recombinant technology, of a Xenopus laevis C-terminal a-amidating enzyme derived having the amino acid sequence set forth in SEQ ID NO: 2 is expressed in E.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 a-amidating enzyine 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 a-amidating enzyine;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.

Its apparent molecular mass (43 kd), estimated by both SDS-PAGE and molecular sieving, was higher than the value (39 kd) for the ' PAM' (AE-I) purified from frog skin.

N-terminal sequence analysis indicated that cleavage of signal sequence had occurred but the propeptide still remained at the N terminus.

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[2] The C-terminal a-amidating enzyme derivative according to the above [1] wherein a disulfide bond has been formed between the cysteine residues at positions 73 and 90 and between the cysteine residues at positions 186 and 293 in the amino acid sequence set forth in SEQ IDNO: 2.