Research Repository

See what's under the surface

Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris

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

Authors

Graham Whyteside

Marcos J.C. Alcocer

Janet R. Kumita

Christopher M. Dobson

Maria Lazarou

Richard J. Pleass

David B. Archer

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.

Journal Article Type Article
Publication Date Jul 27, 2011
Journal PLoS ONE
Electronic ISSN 1932-6203
Publisher Public Library of Science
Peer Reviewed Peer Reviewed
Volume 6
Issue 7
Article Number e22692
Institution Citation Whyteside, G., Alcocer, M. J., Kumita, J. R., Dobson, C. M., Lazarou, M., Pleass, R. J., & Archer, D. B. (2011). Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris. PLoS ONE, 6(7), doi:10.1371/journal.pone.0022692
DOI https://doi.org/10.1371/journal.pone.0022692
Publisher URL http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0022692
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

Files

David__Archer--Native-state.pdf (497 Kb)
PDF

Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0



Downloadable Citations