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Secretion and expression dynamics of a GFP-tagged mucin-type fusion protein in high cell density Pichia pastoris bioreactor cultivations

Journal article
Authors Magnus Sjöblom
Linda Lindberg
Jan Holgersson
Ulrika Rova
Published in Advances in Bioscience and Biotechnology
Volume 3
Issue 3
Pages 238-248
ISSN 2156-8456
Publication year 2012
Published at Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine
Pages 238-248
Language en
Links dx.doi.org/10.4236/abb.2012.33033
Keywords GFP, GFP-Fusion, KAR2, Pichia pastoris, secretion, unfolded protein response, yeast
Subject categories Other Medical Biotechnology

Abstract

The methanol inducible alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-factor prepro secretion signal were used to drive expression and secretion of a mucin-type fusion protein by Pichia pastoris in 1 L scale bioreactors. The aim of the study was to understand how varying expression rates influenced the secretion dynamics of the fusion protein in terms of intracellular- and extracellular concentrations. Endoplasmic reticulum (ER) folding stress was assessed by the relative expression of the unfolded protein response controlled KAR2 gene. Three predefined methanol feeding models were applied to control the fusion protein synthesis rate. To track the fusion protein synthesis in a non-invasive manner and to follow its intracellular distribution, its C-terminal was linked to the green fluorescent protein. Under all conditions the fusion protein was found to partially accumulate intracellularly, where the major fraction was an insoluble, fluorescent full-sized protein. The high degree of glycosylation of the insoluble fusion protein indicated a secretory bottle-neck in the Golgi-system. This result was consistent with low ER folding stress as quantified by the relative expression of the KAR2 gene. Reduction of recombinant protein synthesis rate, by using lower feed rates of methanol, enhanced extracellular concentrations from 8 to 18 mg·L–1 and reduced the rate of intracellular accumulation. This clearly demonstrates the importance of tuning the synthesis rate with secretory bottle-necks to maintain secretion.

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