A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

<p>Abstract</p> <p>Background</p> <p>Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in <it>Escherichia coli </it>in simple shaken cultures.</p> <p>Results</p> <p>The enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD₆₀₀~10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l^-1cell dry weight could be achieved with the developed technique. In comparison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins.</p> <p>Conclusions</p> <p>We have demonstrated that by applying the novel EnBase^®Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approaches.</p

Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system

<p>Abstract</p> <p>Background</p> <p>Here we describe a new technical solution for optimization of <it>Pichia pastoris </it>shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in <it>P. pastoris </it>is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in <it>P. pastoris </it>is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.</p> <p>Results</p> <p>By applying on-line pO₂monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles.</p> <p>By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels.</p> <p>Conclusion</p> <p>The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.</p

Cultivation in shake flasks with methanol feeding without (A, B) or with (C, D) manual pH adjustment

Cultivations were performed in two phases: initial batch phase in BMG-medium and fed-batch in BMM-medium. Dissolved oxygen (pO) and pH were measured with a wireless measuring system, cell growth was followed by measurement of the OD. Vertical lines represent methanol feeding points in A and C or start of methanol feed in B and D.<p><b>Copyright information:</b></p><p>Taken from "Improved production of human type II procollagen in the yeast in shake flasks by a wireless-controlled fed-batch system"</p><p>http://www.biomedcentral.com/1472-6750/8/33</p><p>BMC Biotechnology 2008;8():33-33.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2315644.</p><p></p

Growth parameters (A) and concentrations of product-related mRNA species (B) during cultivation of a for production of recombinant human collagen II in shake flasks

Cultivation procedures with two methanol pulses per day (control, blue open circles, interrupted blue line) and pO-dependent manual feeding of methanol (red filled circles, red continuous line) were compared.<p><b>Copyright information:</b></p><p>Taken from "Improved production of human type II procollagen in the yeast in shake flasks by a wireless-controlled fed-batch system"</p><p>http://www.biomedcentral.com/1472-6750/8/33</p><p>BMC Biotechnology 2008;8():33-33.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2315644.</p><p></p

Schematic presentation of the wireless data collection and control system for shake flask cultivations

<p><b>Copyright information:</b></p><p>Taken from "Improved production of human type II procollagen in the yeast in shake flasks by a wireless-controlled fed-batch system"</p><p>http://www.biomedcentral.com/1472-6750/8/33</p><p>BMC Biotechnology 2008;8():33-33.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2315644.</p><p></p

SDS-PAGE analysis of expression of human collagen II in shake flask cultivation (experiment 3, Table 1) after 21, 46 and 72 hours cultivation

R represents a reference culture with pulse feeding of methanol and F predetermined quasi-continuous feed. Collagen chains were derived from correctly folded collagen II molecules by HCl-extraction and pepsin digestion. The collagen II chains are marked with an arrow.<p><b>Copyright information:</b></p><p>Taken from "Improved production of human type II procollagen in the yeast in shake flasks by a wireless-controlled fed-batch system"</p><p>http://www.biomedcentral.com/1472-6750/8/33</p><p>BMC Biotechnology 2008;8():33-33.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2315644.</p><p></p

MRNA levels of a) procollagen II chain, b) AOX1, c) translation factor EF3, and d) C-P4H α(I) subunit during shake flask cultivation of

Predetermined quasi-continuous feed of methanol was investigated. The cells were first grown in BMG medium and changed to BMM medium at 0 h. The first sample represents the time when the methanol feeding was started (13 h). Predetermined constant feed (red filled circles); reference culture with pulse Feeding (open blue circles). The data are from experiment 4 in Table 1.<p><b>Copyright information:</b></p><p>Taken from "Improved production of human type II procollagen in the yeast in shake flasks by a wireless-controlled fed-batch system"</p><p>http://www.biomedcentral.com/1472-6750/8/33</p><p>BMC Biotechnology 2008;8():33-33.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2315644.</p><p></p

Swarming, defensive and hygienic behaviour in honey bee colonies of different genetic origin in a pan-European experiment

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Population dynamics of European honey bee genotypes under different environmental conditions

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