Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris.
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.
Prabha
Lakshmi
L
Biocon Limited, Research and Development, 20th KM, Hosur Road, Electronic City P.O., Bangalore 560100, India.
Govindappa
Nagaraj
N
Adhikary
Laxmi
L
Melarkode
Ramakrishnan
R
Sastry
Kedarnath
K
D000595:Amino Acid Sequence; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000077270:Exenatide; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010843:Pichia; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014688:Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D010455:Peptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014688:Venoms; D000077270:Exenatide; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C061833:STE13 protein, S cerevisiae