Unigene Laboratories Inc.

Barry N Jones

James P Gilligan

Nozer M Mehta

Gary Agide Beaudry

Arthur H Bertelsen

Purified enzymatic compositions are provided having alpha-amidating enzymes capable of catalyzing the conversion of a peptidyl compound having a C-terminal glycine residue to a corresponding peptidyl amide having an amino group in place of the C-terminal glycine. The purified compositions have specific activities above 25 mU per mg protein and are sufficiently free of proteases to allow effective catalysis of even peptidyl compounds having L-amino acids. Biologically important alpha-amidated products such as calcitonin and other regulatory hormones are efficiently produced using the alpha-amidation reaction catalyzed by the enzymes. Purification by size exclusion chromatography in combination with strong anion exchange chromatography results in homogeneous enzyme species which are used to prepare antibodies specific for the alpha-amidating enzyme. A gene capable of expressing the alpha-amidating enzyme is ligated into an expression vector and transformed into a host cell capable of expressing the gene.

Alpha-amidating enzyme compositions and processes for their production and use

Alpha-amidating enzyme is produced by recombinant DNA techniques recoverable in high yields and at high purity. Both eukaryotic and prokaryotic expression vectors are provided having a transcriptional promoter followed downstream by a DNA sequence which encodes amidating enzyme. The vector selected is one capable of directing the expression of polypeptides in the host selected, and preferred hosts are transected with the described vectors.

Expression systems for amidating enzyme

Peptidyl-glycine .alpha.-amidating monooxygenase is an enzyme extractable from medullary thyroid carcinoma cell lines and tissue samples, having a molecular mass of about 60,000 to 65,000 daltons. It has been purified so as to exhibit a single, homogeneous, well-defined band using electrophoretic procedures performed on SDS-polyacrylamide gels, and has a specific enzymatic activity of at least 50 mU per mg protein. The free or immobilized enzyme, in the presence of Cu.sup.+2 ions, ascorbate, and oxygen, can be used to prepare an .alpha.-amidated protein from a polypeptide substrate possessing a carboxyl-terminal glycine residue. The purified enzyme can be used as an antigen in order to produce enzyme-specific monoclonal antibodies, and can provide the information necessary to design and construct prokaryotes or other appropriate unicellular organisms or host cells isolated from multicellular organisms which possess peptidyl-glycine .alpha.-amidating capability.

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West Milford, NJ, United States of America

Barry N Jones