Secretion and expression dynamics of a GFP-tagged mucin-type fusion protein in high cell density &amp;lt;i&amp;gt;Pichia pastoris&amp;lt;/i&amp;gt; bioreactor cultivations

Magnus Sjöblom,Ulrika Rova,Linda Lindberg,Jan Holgersson

doi:10.4236/abb.2012.33033

Secretion and expression dynamics of a GFP-tagged mucin-type fusion protein in high cell density &lt;i&gt;Pichia pastoris&lt;/i&gt; bioreactor cultivations

Magnus Sjöblom, Ulrika Rova + Show 2 more

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https://doi.org/10.4236/abb.2012.33033

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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.

Highlights

The methylotrophic yeast Pichia pastoris is a robust microorganism for industrial cultivation and has been used to express and secrete several recombinant proteins in the g per litre range [1]
Various strategies have been employed to try to improve secretion including the use of different promoters [2], gene copy number [2,4], secretion signals [5,6], overexpression of proteins involved in the folding and secretion processes [7,8], manipulation of genes involved in cellwall biogenesis [9] and different cultivation techniques [10,11]
The homodimeric mucin-type fusion protein consisting of the extracellular part of P-selectin glycoprotein ligand1 and the Fc part of mouse Immunoglobulin G2b (PSGL1/mIgG2b) was used as the model protein

Summary

Introduction

The methylotrophic yeast Pichia pastoris is a robust microorganism for industrial cultivation and has been used to express and secrete several recombinant proteins in the g per litre range [1]. Various strategies have been employed to try to improve secretion including the use of different promoters [2], gene copy number [2,4], secretion signals [5,6], overexpression of proteins involved in the folding and secretion processes [7,8], manipulation of genes involved in cellwall biogenesis [9] and different cultivation techniques [10,11]. The major limitations to secretion in recombinant yeast are signal sequence processing, membrane translocation and folding within ER [2]. Proteolytic degradation of the product in the media is another factor which often hampers high cell density cultivations and may reduce absolute yields [12,13]

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