Cell lines for expressing enzyme useful in the preparation of amidated products

摘要

Cell lines are provided for expressing peptidylglycine alpha-amidating monooxygenase (PAM), or one of its two catalytic domains. High levels of enzyme expression are achieved while utilizing a non-animal source, low protein tissue culture medium. A robust two-step downstream purification results in high enzyme purity. Resulting PAM, or its PHM catalytic domain, is used to catalyze the enzymatic conversion of X-Gly to X-alpha-hydroxy-Gly or X—NH2 (X being a peptide or any chemical compound having a carbonyl group to which a glycine group can be covalently attached). Methods of preparing preferred cell lines are also set forth.

主权项

1. A CHO KI cell transfected with an expression vector for expressing peptidylglycine alpha-amidating monooxygenase, wherein said expression vector has a coding region encoding peptidylglycine alpha-amidating monooxygenase, wherein the coding region is operably linked to a control region including a ribosome binding site, a promoter, and an SV40 enhancer upstream of said promoter,
wherein the promoter is an inducible human metallothionein IIa promoter and is located immediately upstream of a start site of the coding region, wherein the SV40 enhancer is located immediately upstream of the inducible human metallothionein IIa promoter, wherein the peptidylglycine alpha-amidating monooxygenase consists of a signal peptide at amino acid residues 1 to 25, a pro-region at amino acid residues 26 to 41, and a mature peptidylglycine alpha-amidating monooxygenase at amino acid residues 42 to 715 as set forth in SEQ ID NO: 2, wherein the CHO K1 cell is capable of expressing a soluble peptidylglycine alpha-amidating monooxygenase.