Abstract
BackgroundThe budding yeast Pichia pastoris is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intracellular protein formation, degradation and secretion defines the major bottleneck of the production system. Because these parameters are different for unlimited growth (shake flask) and carbon-limited growth (bioreactor) conditions, they should be determined under "production like" conditions. Thus labeling procedures must be compatible with minimal production media and the usage of bioreactors. The inorganic and non-radioactive 34S labeled sodium sulfate meets both demands.ResultsWe used a novel labeling method with the stable sulfur isotope 34S, administered as sodium sulfate, which is performed during chemostat culivations. The intra- and extracellular sulfur 32 to 34 ratios of purified recombinant protein, the antibody fragment Fab3H6, are measured by HPLC-ICP-MS. The kinetic model described here is necessary to calculate the kinetic parameters from sulfur ratios of consecutive samples as well as for sensitivity analysis. From the total amount of protein produced intracellularly (143.1 μg g-1 h-1 protein per yeast dry mass and time) about 58% are degraded within the cell, 35% are secreted to the exterior and 7% are inherited to the daughter cells.ConclusionsA novel 34S labeling procedure that enables in vivo quantification of intracellular fluxes of recombinant protein under "production like" conditions is described. Subsequent sensitivity analysis of the fluxes by using MATLAB, indicate the most promising approaches for strain improvement towards increased secretion.
Highlights
The budding yeast Pichia pastoris is widely used for protein production
In this work we present a novel 34S labeling method during chemostat cultivation, providing data to consider intracellular protein formation, intracellular degradation, secretion and dilution by growth in the kinetic model
The dynamics of the intra- and extracellular Fab3H6 pools are expressed in two separate differential equations (1) and (3), which are modified from Noe and Delenick [19] and Batt and Kompala [20]
Summary
To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. The balance between intracellular protein formation, degradation and secretion defines the major bottleneck of the production system. Because these parameters are different for unlimited growth (shake flask) and carbon-limited growth (bioreactor) conditions, they should be determined under “production like” conditions. The production of recombinant proteins in yeast has to compete with other host organisms, mainly bacteria and mammalian cell lines. Yeasts like Pichia pastoris grow faster and to a higher cell density compared to mammalian cells, the low specific productivity (the amount of secreted protein per unit biomass and time) is their major drawback [1]. Examples are Sso and Sso, both coding for plasma membrane tSNARE proteins [17] or Cog, Coy and Bmh, all coding for proteins involved in vesicular transport [18]
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