A re-usable wave bioreactor for protein production in insect cells

Wave-mixed bioreactors have increasingly replaced stainless steel stirred tank reactors in seed inoculum productions and mammalian cell-based process developments. Pre-sterilized, single-use plastic bags are used for cultivation, eliminating the risk of cross-contamination and cleaning procedures. However, these advantages come with high consumable costs which is the main barrier to more uptakes of the technology by academic institutions. As an academic Core Facility that faces high demand in protein production from insect cells, we have therefore developed a cost-effective alternative to disposable wave bags. In our study we identified: A re-usable wave shaken polycarbonate bioreactor for protein production in insect cells achieves protein yields comparable to disposable bags. The advantages of this re-usable bioreactor are low costs, long life cycle, flexible configuration of accessories and convenient handling due to its rigid shape. (C) 2016 Max-Planck Institute of Biochemistry. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

A new method to customize protein expression vectors for fast, efficient and background free parallel cloning

Background: Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector. Results: Here we describe a method to tailor selected expression vectors for parallel Sequence and Ligation Independent Cloning. SLIC cloning enables precise and sequence independent engineering and is based on joining vector and insert with 15-25 bp homologies on both DNA ends by homologous recombination. We modified expression vectors based on pET, pFastBac and pTT backbones for parallel PCR-based cloning and screening in E. coli, insect cells and HEK293E cells, respectively. We introduced the toxic ccdB gene under control of a strong constitutive promoter for counterselection of insert less vector. In contrast to DpnI treatment commonly used to reduce vector background, ccdB used in our vector series is 100% efficient in killing parental vector carrying cells and reduces vector background to zero. In addition, the 3' end of ccdB functions as a primer binding site common to all vectors. The second shared primer binding site is provided by a HRV 3C protease cleavage site located downstream of purification and solubility enhancing tags for tag removal. We have so far generated more than 30 different parallel expression vectors, and successfully cloned and expressed more than 250 genes with this vector series. There is no size restriction for gene insertion, clone efficiency is > 95% with clone numbers up to 200. The procedure is simple, fast, efficient and cost-effective. All expression vectors showed efficient expression of eGFP and different target proteins requested to be produced and purified at our Core Facility services. Conclusion: This new expression vector series allows efficient and cost-effective parallel cloning and thus screening of different protein constructs, tags and expression hosts

A concise guide to choosing suitable gene expression systems for recombinant protein production

This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making. Additionally, information has been included for selected less frequently used "exotic" gene expression systems

Increased Hydrogen Production by Genetic Engineering of Escherichia coli

Escherichia coli is capable of producing hydrogen under anaerobic growth conditions. Formate is converted to hydrogen in the fermenting cell by the formate hydrogenlyase enzyme system. The specific hydrogen yield from glucose was improved by the modification of transcriptional regulators and metabolic enzymes involved in the dissimilation of pyruvate and formate. The engineered E. coli strains ZF1 (ΔfocA; disrupted in a formate transporter gene) and ZF3 (ΔnarL; disrupted in a global transcriptional regulator gene) produced 14.9, and 14.4 µmols of hydrogen/mg of dry cell weight, respectively, compared to 9.8 µmols of hydrogen/mg of dry cell weight generated by wild-type E. coli strain W3110. The molar yield of hydrogen for strain ZF3 was 0.96 mols of hydrogen/mol of glucose, compared to 0.54 mols of hydrogen/mol of glucose for the wild-type E. coli strain. The expression of the global transcriptional regulator protein FNR at levels above natural abundance had a synergistic effect on increasing the hydrogen yield in the ΔfocA genetic background. The modification of global transcriptional regulators to modulate the expression of multiple operons required for the biosynthesis of formate hydrogenlyase represents a practical approach to improve hydrogen production

On-line analysis and in situ pH monitoring of mixed acid fermentation by Escherichia coli using combined FTIR and Raman techniques

We introduce an experimental setup allowing continuous monitoring of bacterial fermentation processes by simultaneous optical density (OD) measurements, long-path FTIR headspace monitoring of CO2, acetaldehyde and ethanol, and liquid Raman spectroscopy of acetate, formate, and phosphate anions, without sampling. We discuss which spectral features are best suited for detection, and how to obtain partial pressures and concentrations by integrations and least squares fitting of spectral features. Noise equivalent detection limits are about 2.6 mM for acetate and 3.6 mM for formate at 5 min integration time, improving to 0.75 mM for acetate and 1.0 mM for formate at 1 h integration. The analytical range extends to at least 1 M with a standard deviation of percentage error of about 8%. The measurement of the anions of the phosphate buffer allows the spectroscopic, in situ determination of the pH of the bacterial suspension via a modified Henderson-Hasselbalch equation in the 6–8 pH range with an accuracy better than 0.1. The 4 m White cell FTIR measurements provide noise equivalent detection limits of 0.21 μbar for acetaldehyde and 0.26 μbar for ethanol in the gas phase, corresponding to 3.2 μM acetaldehyde and 22 μM ethanol in solution, using Henry’s law. The analytical dynamic range exceeds 1 mbar ethanol corresponding to 85 mM in solution. As an application example, the mixed acid fermentation of Escherichia coli is studied. The production of CO2, ethanol, acetaldehyde, acids such as formate and acetate, and the changes in pH are discussed in the context of the mixed acid fermentation pathways. Formate decomposition into CO2 and H2 is found to be governed by a zeroth-order kinetic rate law, showing that adding exogenous formate to a bioreactor with E. coli is expected to have no beneficial effect on the rate of formate decomposition and biohydrogen production

Inducible protein expression in piggyBac transposase mediated stable HEK293 cell pools

Described here is the use of piggyBac transposase generated HEK293 stable cell pools for doxycycline-inducible protein production. The key benefits of the system are that low amounts of plasmid DNA are needed for transfection, high levels of protein expression can be achieved also for toxic proteins at robust scalability and reproducibility and the recombinant cell line can be stored as frozen cell bank. Transfection, selection, expression and purification of enhanced green fluorescence protein (eGFP) and SARS-CoV-2 Spike protein are described in this chapter

A fast-track protocol for protein expression using the BEV system

Baculovirus-insect cell expression (BEV) has become one of the most widely used eukaryotic systems for heterologous protein expression. The combination of engineered baculovirus genomes together with a variety of compatible vectors, robust insect cell lines, serum-free media and commercial kits have made it a standard workhorse in many "non-virology-expert" laboratories. Despite these significant improvements, the BEV system still has major drawbacks, primarily the time required to amplify recombinant virus and its inherent instability. Here we present an easy-to-adopt simplified and shortened protocol