Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris |
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Authors: | Lakshmi Prabha Nagaraj Govindappa Laxmi Adhikary Ramakrishnan Melarkode Kedarnath Sastry |
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Affiliation: | aBiocon Limited, Research and Development, 20th KM, Hosur Road, Electronic City P.O., Bangalore 560 100, India |
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Abstract: | 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. |
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Keywords: | Dipeptidyl aminopeptidase Pichia pastoris Saccharomyces cerevisiae STE13 DAP2 Exendin-4 |
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