Loading…

Secretory expression of bovine trypsinogen by Pichia pastoris utilizing a truncated alpha-factor secretory signal sequence and a modified propeptide sequence

Trypsin plays a crucial role in processing recombinant precursor insulins, especially in removing a peptide linker or a peptide spacer by cleaving the peptide after a lysine or arginine residue. To reduce the cell toxicity of the expressed trypsin and increase the secretory expression, we developed...

Full description

Saved in:
Bibliographic Details
Main Authors: Kusharyoto, Wien, Hariyatun, Hariyatun, Nurdiani, Dini, Putro, Eko Wahyu, Utami, Nuruliawaty, Silvia, Rozy Ayu, Patria, Fadillah Putri
Format: Conference Proceeding
Language:English
Subjects:
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Trypsin plays a crucial role in processing recombinant precursor insulins, especially in removing a peptide linker or a peptide spacer by cleaving the peptide after a lysine or arginine residue. To reduce the cell toxicity of the expressed trypsin and increase the secretory expression, we developed a strategy for producing a recombinant bovine trypsinogen using the methylotrophic yeast Pichia pastoris. The expression cassette contains the gene encoding a truncated α-mating factor from Saccharomyces cerevisiae with a cleavage site for a Kex2 endoprotease, a short propeptide having the cleavage site for autolysis (DDDDK), followed by the sequence for the active trypsin. Furthermore, based on a 3D structure model, we constructed a variant of bovine trypsin, which would have a higher specificity in cleaving a polypeptide after a lysine residue rather than an arginine. Clones resistant to zeocin could be isolated and checked using PCR with primers specific to AOX1 to integrate the expression cassette into the genome of P. pastoris and to identify Mut phenotypes. Trypsinogen secretion into the supernatant was demonstrated using SDS-PAGE, with which a single band of the secreted trypsinogen with a molecular weight of approximately 25 kDa was detected. The method described here represents an initial step in developing "halal" trypsin, particularly for its application in the production of recombinant insulin and its analogs.
ISSN:0094-243X
1551-7616
DOI:10.1063/5.0204757