Handa Sumit, Spradling Tyler J, Dempsey Daniel R, Merkler David J
Department of Chemistry, University of South Florida, Tampa, FL 33620, USA.
Protein Expr Purif. 2012 Jul;84(1):9-13. doi: 10.1016/j.pep.2012.04.012. Epub 2012 Apr 25.
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-His(6) 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.
大多数哺乳动物生物活性肽都具有C端氨基酸酰胺基团。C端酰胺的存在是重组生产α-酰胺化肽的一个重大障碍。α-酰胺化肽在体内是通过对具有C端甘氨酸残基的前体进行酶切产生的。肽基甘氨酸α-羟化单加氧酶催化甘氨酸延伸前体氧化为α-酰胺化肽的关键步骤。在此，我们详细介绍了人肽基甘氨酸α-羟化单加氧酶（hPHMcc）催化核心在大肠杆菌中的生产，该催化核心与硫氧还蛋白（Trx）有N端融合。将Trx与hPHMcc融合，以提高所得52 kDa蛋白作为可溶性且具有催化活性的酶的产量。Trx-hPHMcc-His(6)融合蛋白被纯化至同质，并表现出与纯化的大鼠PHMcc相似的稳态动力学参数。重组hPHMcc的细菌生产将促进通过在大肠杆菌中共表达hPHMcc和α-酰胺化肽前体或对重组表达的α-酰胺化肽前体进行体外酰胺化来生成α-酰胺化肽的努力。
