Fetal bovine serum enhances recombinant protein secretion and stability in Pichia pastoris

Komagataella phaffii, formerly known as Pichia pastoris, is a methylotrophic yeast employed as a recombinant protein factory for academic and industrial purposes. Despite its advantages, some recombinant proteins, which are engineered to be secreted, are retained intracellularly and subject to degradation. Serendipitously, we discovered that fetal bovine serum (FBS) improved the secretion of several proteins. When a P. pastoris strain expressing enhanced green fluorescent protein (eGFP) was grown with FBS, western analysis revealed a 2-3 fold increased level of eGFP secretion as well as the induction of an upper molecular band (30 kD) in addition to the expected band (26.8 kD). FBS protected the C-terminus of eGFP from degradation, and the alterations to this upper molecular weight variant were triggered intracellularly by an FBS component that was most likely not a protein.

To elucidate the mechanisms behind the production of the novel recombinant protein variant, we utilized site-directed mutagenesis and mass spectrometry.

Through these strategies, we were able to localize posttranslational modifications to the C-terminus associated with FBS treatment. Analysis of the intracellular lysate revealed proteomic alterations, suggesting that genes involved in translation, trafficking, stress response and energetics were key players in FBS's effect on the P. pastoris secretion mechanism through interaction with eGFP's C-terminus.

Our study suggests that C-terminal degradation may affect other recombinant proteins produced in P. pastoris, a problem that may be resolved by FBS.