Open AccessConstruction of a full length α-factor secretory signal sequence for human insulin precursor expression in Pichia pastoris
Author(s) -
Ning Wikan Utami,
Dini Nurdiani,
Hariyatun,
Eko Wahyu Putro,
Wien Kusharyoto
Publication year - 2021
Publication title -
iop conference series. earth and environmental science
Language(s) - English
Resource type - Journals
eISSN - 1755-1307
pISSN - 1755-1315
DOI - 10.1088/1755-1315/762/1/012066
Subject(s) - pichia pastoris , expression vector , signal peptide , biology , saccharomyces cerevisiae , electroporation , pichia , cloning (programming) , recombinant dna , microbiology and biotechnology , yeast , genetics , gene , computer science , programming language
Saccharomyces cerevisiae and Pichia pastoris are yeast known as a potential expression system to produce recombinant protein. The full-length α-factor (α-mating factor) secretory signal of S. cerevisiae plays an essential role in the secretion and processing of the mature protein of interest. Here, we attempted to construct the full-length α-factor signal sequence of S. cerevisiae in the pD902-IP (Insulin Precursor) expression vector for secreted expression of human insulin precursor in P. pastoris . We have isolated the full-length α-factor secretory signal sequence in a pTA2 cloning vector. The full-length α-factor was then inserted into the IP-cassette of pD902-IP and transformed into E. coli TOP10. The E. coli transformants, which were able to grow on the Zeocin selection medium, harbored the full-length α-factor for the IP expression in the pD902 vector validated by PCR and sequencing. Furthermore, the construct electroporation into P. pastoris X-33 was done and followed by IP protein expression confirmation visualized with SDS-PAGE.