Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures

BACKGROUND: A temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut(+ )strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain. RESULTS: A protein concentration of about 1 g L(-1 )was produced in the MLFB process. However, this product was considerably degraded by protease(s). By applying the TLFB process, the yield was increased to 2 g L(-1 )full-length product and no proteolytic degradation was observed. Flow cytometry analysis showed that the percentage of dead cells increased rapidly during the initial methanol feed phase in the MLFB process and reached a maximum of about 12% after about 40–70 hours of methanol feeding. In the TLFB process, cell death rate was low and constant and reached 4% dead cells at the end of cultivation (about 150 hours methanol feeding time). The lower cell death rate in the TLFB correlated with a lower protease activity in the culture supernatant. The specific alcohol oxidase (AOX) activity in the TLFB process was 3.5 times higher than in the MLFB process. CONCLUSION: Three mechanisms that may contribute to the much higher accumulation of product in the TLFB process are: 1) reduced proteolysis due to lower temperature, 2) reduced proteolysis due to lower cell death and protease release to the medium, 3) increased synthesis rate due to higher AOX activity

On the measurement of subsonic flow in a capacitively coupled helicon plasma source

Plasma parameters and the subsonic flow from a capacitively coupled, cylindrical plasma source of the Njord helicon device are investigated by means of a Mach probe and a retarding field energy analyzer (RFEA). 13.56 MHz and 600 W RF power is inserted into the argon working gas under low-pressure conditions and moderate magnetic field. By means of a downstream field coil, the magnetic field is shaped from a purely expanding field to a configuration with more parallel field lines. It is shown that the downstream plasma density along the outer rim of the source increases significantly and there is a sudden increase by nearly 20 V in the plasma potential already after a moderate increase in the downstream magnetic field. The investigation of the flow indicates that current ratios derived from the Mach probe result in an apparent flow in the direction towards the source, while the current bratios derived from the RFEA indicate a flow in the direction away from the source. PIC simulations demonstrate that the acceptance angle of the probes, being nearly 180o for the Mach probe, and about 45o for the RFEA, can critically affect the current ratios and hence the subsonic flow measured by the probes in the weakly magnetized plasma in our device. The first section in your pape

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

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

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