Native-State Stability Determines the Extent of Degradation Relative to Secretion of Protein Variants from Pichia pastoris

Graham Whyteside,Janet R Kumita,Richard J Pleass,David B Archer,Marcos J C Alcocer,Maria Lazarou,Christopher M Dobson,Christopher B Doering

doi:10.1371/journal.pone.0022692

Graham Whyteside, Janet R Kumita + Show 6 more

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https://doi.org/10.1371/journal.pone.0022692

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Abstract

We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general.

Highlights

Yeasts have become increasingly common hosts for the expression of eukaryotic heterologous proteins due to their ease of culture and genetic manipulation, well defined fermentation processes and rapid growth to high cell densities
To test this hypothesis the nine human lysozyme (HuL) variants were expressed in P. pastoris for 48h and the mRNA levels of the unfolded protein response (UPR) marker genes HAC1, KAR2 and PDI1 were assessed at 6, 12, 24 and 48 h using qRT-PCR
It is logical to postulate that the variantdependent increase in levels of HAC1 mRNA observed in Figure 1A would lead to an increase in UPR activation

Summary

Introduction

Yeasts have become increasingly common hosts for the expression of eukaryotic heterologous proteins due to their ease of culture and genetic manipulation, well defined fermentation processes and rapid growth to high cell densities. These advantages have led to a number of studies concerning the optimisation of yeast as cell factories for the secretion of heterologous proteins that include therapeutic proteins [1,2]. P. pastoris has many advantages over S. cerevisiae including growing to higher cell densities, the availability of strong and tightly controlled promoters and having a low immunogenic glycosylation pattern [5] These advantages combined with the recently published genome sequence [6,7] of this organism have made P. pastoris the yeast expression system of choice for many researchers

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