کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2020587 | 1069193 | 2012 | 5 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli](/preview/png/2020587.png)
Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His6 fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors.
► Expression of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in E. coli.
► Production of soluble protein.
► Catalytically active hPHMcc.
Journal: Protein Expression and Purification - Volume 84, Issue 1, July 2012, Pages 9–13