Antje Neubauer

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

BackgroundCultivations 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 Escherichia coli in simple shaken cultures.ResultsThe 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-1 cell 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.ConclusionsWe 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.

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

BackgroundHere we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris 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 P. pastoris 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.ResultsBy applying on-line pO2 monitoring 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.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.ConclusionThe 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.

Mini-scale cultivation method enables expeditious plasmid production in Escherichia coli

The standard procedure in the lab for plasmid isolation usually involves a 2‐mL, 16 h over‐night cultivation in 15‐mL bioreaction tubes in LB medium. This is time consuming, and not suitable for high‐throughput applications. This study shows that it is possible to produce plasmid DNA (pDNA) in a 1.5‐mL microcentrifuge tube with only 100 μL cultivation volume in less than 7 h with a simple protocol. Compared with the standard LB cultivation for pDNA production reaching a final pDNA concentration range of 1.5–4 μg mL–1, a 6‐ to 10‐fold increase in plasmid concentration (from 10 up to 25 μg mL–1 cultivation volume) is achieved using an optimized medium with an internal substrate delivery system (EnBase®). Different strains, plasmids, and the applicability of different inoculation tools (i.e. different starting ODs) were compared, demonstrating the robustness of the system. Additionally, dissolved oxygen was monitored in real time online, indicating that under optimized conditions oxygen limitation can be avoided. We developed a simple protocol with a significantly decreased procedure time, enabling simultaneous handling of more samples, while a consistent quality and a higher final pDNA concentration are ensured.

The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli.

While the nutrient limited fed-batch technology is the standard of the cultivation of microorganisms and production of heterologous proteins in industry, despite its advantages in view of metabolic control and high cell density growth, shaken batch cultures are still the standard for protein production and expression screening in molecular biology and biochemistry laboratories. This is due to the difficulty and expenses to apply a controlled continuous glucose feed to shaken cultures. New ready-made growth media, e.g. by biocatalytic release of glucose from a polymer, offer a simple solution for the application of the fed-batch principle in shaken plate and flask cultures. Their wider use has shown that the controlled diet not only provides a solution to obtain significantly higher cell yields, but also in many cases folding of the target protein is improved by the applied lower growth rates; i.e. final volumetric yields for the active protein can be a multiple of what is obtained in complex medium cultures. The combination of the conventional optimization approaches with new and easy applicable growth systems has revolutionized recombinant protein production in Escherichia coli in view of product yield, culture robustness as well as significantly increased cell densities. This technical development establishes the basis for successful miniaturization and parallelization which is now an important tool for synthetic biology and protein engineering approaches. This review provides an overview of the recent developments, results and applications of advanced growth systems which use a controlled glucose release as substrate supply.

Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase

BackgroundWe describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α2β2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.ResultsWe applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase®) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield.ConclusionsHere we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits.

Toward Microbioreactor Arrays: A Slow-Responding Oxygen Sensor for Monitoring of Microbial Cultures in Standard 96-Well Plates.

In this study, a slow-responding chemo-optical sensor for dissolved oxygen (DO) integrated into a 96-well plate was developed. The slow response time ensures that the measured oxygen value does not change much during plate transport to the microplate reader. The sensor therefore permits at-line DO measurement of microbial cultures. Moreover, it eliminates the necessity of individual optical measurement systems for each culture plate, as many plates can be measured successively. Combined with the 96-well format, this increases the experimental throughput enormously. The novel sensor plate (Slow OxoPlate) consists of fluorophores suspended in a polymer matrix that were placed into u-bottom 96-well plates. Response time was measured using sodium sulfite, and a t90 value of 9.7 min was recorded. For application, DO values were then measured in Escherichia coli and Saccharomyces cerevisiae cultures grown under fed-batch–like conditions. Depending on the DO sensor’s response time, different information on the oxygenation state of the culture plate was obtained: a fast sensor variant detects disturbance through sampling, whereas the slow sensor indicates oxygen limitation during incubation. A combination of the commercially available OxoPlate and the Slow OxoPlate enables operators of screening facilities to validate their cultivation procedures with regard to oxygen availability.

High Yield of Recombinant Protein in Shaken E. coli Cultures with Enzymatic Glucose Release Medium EnPresso B

Expression of recombinant proteins in sufficient quantities is essential for protein structure-function studies. The most commonly used method for recombinant protein production is overexpression in E. coli cultures. However, producing high yields of functional proteins in E. coli can be a challenge in conventional shaken cultures. This is often due to nonoptimal growth conditions, which result in low cell yields and insoluble or incorrectly folded target protein. To overcome the shortcomings of shake flask cultivation, we present a cultivation method based on enzymatic glucose delivery. This system mimics the fed-batch principle used in bioreactor cultivations and provides high yields of biomass and recombinant proteins in shaken cultivations.

Optimisation of substrate feeding in shake flask cultures of Pichia pastoris for recombinant protein production

who generously supported the meeting. Meeting abstracts - A single PDF containing all abstracts in this supplement is available he re . http://www. biomedcentral.co m/content/pdf/14 75-2859-5-S1-inf o.pdf

Use of the tetA-promoter in fed-batch cultivations: Repeated supply of anhydrotetracycline is necessary for production of tetrameric collagen prolyl 4-hydroxylase in Escherichia coli

Background Human collagen prolyl 4-hydroxylase (C-P4H), an ER lumenal protein, is a key enzyme in the biosynthesis of collagens and consists of two different subunits forming an α2β2 tetramer. Heterologous cytoplasmic production of an active C-P4H in Escherichia coli using a bicistronic vector with the T5-lac and tet promoters and the strain OrigamiTM as a host was described earlier [1]. Gene optimisation of the β subunit that is identical to protein disulfide isomerase and selection of the best induction conditions improved the obtained activity of recombinant C-P4H in shake flask cultivations further by a factor of 50 [2].