Construction of a full length α-factor secretory signal sequence for human insulin precursor expression in Pichia pastoris

Abstract

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.