Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris

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• 作者：Lakshmi Prabha ; Nagaraj Govindappa ; Laxmi Adhikary ; Ramakrishnan Melarkode ; Kedarnath Sastry
• 关键词：Dipeptidyl aminopeptidase ; Pichia pastoris ; Saccharomyces cerevisiae ; STE13 ; DAP2 ; Exendin-4
• 刊名：Protein Expression and Purification
• 出版年：2009
• 出版时间：April 2009
• 年：2009
• 卷：64
• 期：2
• 页码：155-161
• 全文大小：1752 K

文摘

Exendin-4 is a naturally occurring 39 amino acid peptide that is useful for the control of Type 2 diabetes. Recombinant Exendin-4, with an extra glycine at the carboxy-terminus (Exdgly), was expressed in the methylotropic yeast Pichia pastoris. A high proportion of the Exdgly molecules secreted into medium were found to be clipped, lacking the first two amino acids (His–Gly) from the N-terminus. Disruption of the P. pastoris homolog of the Saccharomyces cerevisiae dipeptidyl aminopeptidase (STE13) gene in Pichia genome resulted in a clone that expressed N-terminally intact Exdgly. Elimination of N-terminal clipping enhanced the yield and simplified the purification of Exdgly from P. pastoris culture supernatant.