High‐Level Expression and Purification of Tag‐free Peptides Containing Multiple Disulfide Bond in Pichia pastoris

Abstract

Eukaryotic expression systems are used widely and have the advantages of protein processing, proteolytic cleavage, disulfide bond formation, and posttranslational modification in contrast to the prokaryotic expression system. In the present study, peptide gene (olive flounder beta‐defensin or hepcidin) was inserted into the vector of pPIC9K, which involved the secretion signal and promoter AOX1. The colonies with high copy numbers of the target gene for high‐level expression were selected by G418. Approximately 30 mg/L for beta‐defensin and 25 mg/L for hepcidin was obtained from the culture medium supernatant. An ammonium sulfate salting‐out method was used for purification; this one‐step purification simplified the procedures, and the purification effect was good in terms of the purity and yield. The proteins from yeast itself could be isolated easily using the ammonium sulfate salting‐out method.