Optimisation and evaluation of a high-throughput mammalian protein expression system

doi:10.1016/j.pep.2005.03.012

Protein Expression and Purification

Volume 42, Issue 1, July 2005, Pages 111-121

https://doi.org/10.1016/j.pep.2005.03.012 Get rights and content

Abstract

Transient transfection of mammalian cells with episomal vectors is a very useful method for producing high levels of recombinant proteins. Transient systems remove the need for the laborious and time-consuming process of creating stable cell lines. Here, we describe the optimisation and evaluation of a high-throughput transient expression system in HEK293-EBNA cells. The process was developed for the expression of 10 constructs simultaneously in deep-well plates and subsequent purification using 96-well plate affinity chromatography. This enabled multiple combinations of different constructs, vectors, and expression conditions to be studied in parallel.

Section snippets

GATEWAY destination vectors

The destination vectors pCEP4-GW, pCEP46His-GW, pCEP4flag-GW, pCEP4C6His-GW, and pCEP4Cflag-GW were all modified in-house from pCEP4 (Invitrogen) using the Gateway Technology kit (www.Invitrogen.com) (catalogue Nos. 12535-019 and 12535-027). The destination vector pEAK10MCS-GW was modified from pEAK10 (Edge Biosystems). AmCyan was obtained from Clontech.

Cloning and subcloning

The genes used for this study were obtained from our in-house collections for vector constructs. They had been sequence verified in either

Results and discussion

Transient gene expression in HEK293-EBNA suspension cells with episomal expression vectors has been reported by a number of groups [10], [11], [20]. The challenge for the use of this expression system in a high-throughput parallel manner was the miniaturisation of the method to enable a quick and economic process. Presented here are a series of experiments that were carried out to optimise and evaluate the transfection process in 24 deep-well plates and subsequent affinity purification in a

Summary

Here, we have described a transient mammalian expression platform that has been adapted for high-throughput transfection and purification of recombinant proteins. This is a rapid, cost effective, and scaleable method that will be of great use as a screening strategy to decide which proteins to express at larger scale in HEK293-EBNA cells. We have shown that the method was robust and reproducible when used to screen the expression of both intracellular and secreted proteins, and has been

Acknowledgments

We thank Chloe Tyler and Dawood Dassu for their support with cell culture and statistical analysis of FED data, respectively. We would like to thank Lisa Evans and Eelco Docter for the GATEWAY adaptation of pCEP4. We also thank Paul Hawtin and Ian Hardern for their help with the Biorobot 8000. We also thank Ian Taylor for critical appraisal of the manuscript and Wolfgang Knecht for his collaboration with ProCPU. We also acknowledge the kind help of Tanja Wulff from Agilent Technologies in

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Citation Excerpt :
Mammalian expression systems allow production of active recombinant proteins that overcome these limitations. High level transient expression of recombinant proteins in mammalian systems has been accomplished through the use of plasmids with the capability for episomal replication (Durocher et al., 2002; Davies et al., 2005). A plasmid mediated HTP transient expression of phage-derived antibodies in IgG format was developed for antibody HTP screening (Jostock et al., 2004).
We describe herein a method to enable high throughput (HTP) screening of libraries of soluble proteins such as phage-derived clones of IgG, scFv-Fc, or other Fc-fusion proteins expressed in mammalian cells via adenovirus transduction. DNA fragments of antibody single chains (scFvs) and fragment antigen-binding (Fabs) from the positive clones of the third round of bacteriophage panning against a target antigen were batch reformatted into scFv-Fc or IgG in an oriP bearing entry vector and then recombined to an adenovirus vector through Gateway technology. The resulting antibody gene-containing adenovirus libraries were added to 96-well plates seeded with mammalian cells at a ratio of 0.7 infectious viral particles per well to establish clonality. Protocol optimization improved the expression of scFv-Fc and IgGs up to 100 μg/mL in 96-well plates, which is sufficient for most antibody characterizations. In addition, 78% of the wells that were positive for protein expression contain only one sequence, indicating successful establishment of clonality in a majority of wells. We have established and optimized a mammalian expression system that produces soluble protein variants in a HTP manner. The system will facilitate developing multiple downstream screening methodologies.
A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications
2011, Methods
Citation Excerpt :
As ThinCert™ 6 deep-well plates can be stacked easily and can accommodate up to 8 mL of culture/well, we found them convenient for producing significant quantities of proteins in a high-throughput manner. The method can even be adapted to 24 deep-well plates since transfection of 293-EBNA1 cells grown in these plates was shown to be efficient [36,37]. Moreover, we have shown that plasmid DNA prepared using silica-based commercial miniprep kits is fully compatible with PEI-mediated transfection, giving expression levels only slightly lower to those obtained with anion-exchange purified plasmids.
Transient gene expression in mammalian cells is a valuable alternative to stable cell lines for the rapid production of large amounts of recombinant proteins. While the establishment of stable cell lines takes 2–6 months, milligram amounts of protein can be obtained within a week following transfection. The polycation polyethylenimine (PEI) is one of the most utilized reagents for small- to large-scale transfections as it is simple to use and, when combined with optimized expression vectors and cell lines, provides high transfection efficiency and titers. As with most transfection reagents, PEI-mediated transfection involves the formation of nanoparticles (polyplexes) which are obtained by its mixing with plasmid DNA. A short incubation period that allows polyplexes to reach their optimal size is performed prior to their addition to the culture. As the quality of polyplexes directly impacts transfection efficiency and productivity, their formation complicates scalability and automation of the process, especially when performed in large-scale bioreactors or small-scale high-throughput formats. To avoid variations in transfection efficiency and productivity that arise from polyplexes formation step, we have optimized the conditions for their creation directly in the culture by the consecutive addition of DNA and PEI. This simplified approach is directly transferable from suspension cultures grown in 6-well plates to shaker flasks and 5-L WAVE bioreactors. As it minimizes the number of steps and does not require an incubation period for polyplex formation, it is also suitable for automation using static cultures in 96-well plates. This “direct” transfection method thus provides a robust platform for both high-throughput expression and large-scale production of recombinant proteins.
Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery
2011, Protein Expression and Purification
Citation Excerpt :
The 400 ng showed the best result in the optimal cell density, 50 k cells/well. This DNA concentration was higher than in the previously published study [18], but the effect might be due to the different growth media and transfection reagents. Similarly, 400 ng/well gave the best result in the higher cell densities, but the absolute protein concentration was lower.
Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a ‘tag after stop codon’ system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS–PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels.
In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity.
HaloTag-based purification of functional human kinases from mammalian cells
2011, Protein Expression and Purification
Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His₆Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.
Recent advances in the production of proteins in insect and mammalian cells for structural biology
2010, Journal of Structural Biology
The production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems.
Modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance
2010, Journal of Chromatography A
Citation Excerpt :
Process design and optimization are critical tasks in any pharmaceutical industrial process; the time and resources allotted for R&D tasks are relevant issues [1,2].
Surface plasmon resonance (SPR) spectroscopy is used as a scaled-down, analytical, pseudo-chromatography tool for analyzing protein binding and elution over an ion-exchange surface under cyclic sorption conditions. A micrometric-scale adsorption surface was produced by immobilizing a typical ion exchange ligand – diethylaminoethyl (DEAE) – onto commercially available planar gold sensor chip surfaces pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid with known density. An explicit mathematical formulation is provided for the deconvolution and interpretation of the SPR sensorgrams. An adsorption rate model is proposed to describe the SPR sensorgrams for bovine serum albumin, used here as model protein, when the DEAE surface is subjected to a cyclic series of binding and elution steps. Overall, we demonstrate that the adsorption rate model is capable of quantitatively describing BSA binding and elution for protein titers from dilute conditions up to overloaded conditions and a broad range of salt concentrations.

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Protein Expression and Purification

Abstract

Section snippets

GATEWAY destination vectors

Cloning and subcloning

Results and discussion

Summary

Acknowledgments

References (22)

Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems

Protein Expr. Purif.

High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells

Protein Expr. Purif.

Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein

J. Biol. Chem.

New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues

J. Chromatogr.

Transfection of partially purified plasmid DNA for high level transient protein expression in HEK293-EBNA cells

J. Biotechnol.

Combinatorial gene expression using multiple episomal vectors

Gene

Large-scale transient expression in mammalian cells for recombinant protein production

Curr. Opin. Biotechnol.

An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography

BMC Biotechnol.

Facilities and methods for the high-throughput crystal structural analysis of human proteins

Acc. Chem. Res.

Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP

Proc. Natl. Acad. Sci. USA

GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes

Methods Enzymol.

Cited by (27)

A mammalian expression system for high throughput antibody screening

A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery

HaloTag-based purification of functional human kinases from mammalian cells

Recent advances in the production of proteins in insect and mammalian cells for structural biology

Modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance