pPIC9k vector

Irisin is a novel hormone implicated in many metabolic diseases. In order to elucidate the functions and therapeutic effects of irisin, it is necessary to obtain active irisin. Human FNDC5 contains 212 amino acids, and the hormone irisin it releases contains 112 amino acids. Interestingly, irisin is completely conserved between mice and humans, sharing 100% primary protein structure. The native form of irisin is likely glycosylated, and the human irisin protein has two putative N-glycosylation sites at asparagine 7 (Asn7) and asparagine 52 (Asn52). In order to obtain active irisin for further research, the recombinant protein irisin was expressed using the Pichia pastoris eukaryotic expression system.

The powerful Pichia pastoris/pPIC9K eukaryotic expression system enables rapid growth at high density. The pPIC9K expression vector carries a strong alcohol oxidase I (AOX1) promoter and α factor signal sequence to help the recombinant protein be secreted out of the cell. Although the final yield of a protein is largely influenced by its intrinsic properties, yields can be significantly improved by manipulating factors that influence gene expression and production stability. In this study, a codon-optimized irisin gene was designed based on the synonymous codon usage preference of Pichia pastoris and cloned into the pPIC9K expression vector. Expression of codon-optimized recombinant irisin in Pichia pastoris GS115. SDS-PAGE and Western blot were used to evaluate the level and purity of irisin. The results showed that the recombinant Pichia pastoris GS115 strain secreted a large amount of irisin into the culture supernatant, but few other proteins. Optimization experiments showed that the yield of recombinant irisin was highest when induced with pH 6.0 and 2.0% methanol for 96 hours, and the concentration of recombinant irisin in the supernatant after codon optimization could reach nearly 77.98 mg/l.

Figure 1. Schematic representation of the pPIC9K-irisin plasmid. (Duan H, et al., 2015)