A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Summary

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Abstract

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) ^1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells ³, and cell-free in vitro translation systems ^2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins ^5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.

Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein ^5,10.

In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO₂ incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

Protocol

1. Preparation Work - Constructs and Cell Cultures

Before starting the protocol, the gene of interest should be codon-optimized for expression in mammalian cells, and cloned into an appropriate expression vector using standard molecular biology techniques. In order to ensure the highest chance for successful expression, multiple variants of the gene of interest should be generated. Many mammalian expression vectors are available commercially and have various purification tags (polyhistidine, hemagglutinin, streptavidin, HALO-Tag, glutathione S-transferase, among others). We prefer to use the pDISPLAY vector, which encodes for a strong human c....

Discussion

The 10-layer cell factories are an effective vessel for the production of milligram quantities of protein. A major advantage of using the cell factory over other traditional vessels, such as roller bottles, shake flasks or spinner flasks, is that they do not require the purchase of any additional laboratory equipment. A standard CO₂ incubator (~6.0 cu. ft.) will easily accommodate four 10-layer cell factories (Fig. 2). In addition, these vessels require less labor and space than dishes, flasks.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alkaline phosphatase (BCIP/NBT) liquid substrate solution	Sigma	B6404
Antibiotic/Antimycotic, 100X	Invitrogen	15240062
Anti-HA affinity matrix, clone 3F10	Roche	1815016
Anti-HA murine mAb, clone 16B12	Covance	MMS-101P
Cell culture flask, T75 cm2 tissue culture treated	Corning	430641
Cell culture flask, T225 cm2 tissue culture treated	Corning	431082
Cell culture plates,6-well tissue culture treated	Corning	3516
Cell factory, 10-layer CellSTACK	Corning	3312
Centramate Omega 5K Cassette	Pall	OS005C12
Centramate Omega 30K Cassette	Pall	OS030C12
Chromatography glass column, 1.0x10 cm	Kontes	4204001010
Ciprofloxacin	Sigma	17850
CO2
Dulbecco's modified Eagle's media (DMEM)	Sigma	D5796
Fetal bovine serum (FBS), heat inactivated	Invitrogen	12484-028
FuGENE HD transfection reagent	Promega	4709691001
GeneJuice transfection reagent	EMD/Merck	70967-6
Glycine	Sigma	G8898
Goat anti-mouse IgG F(ab')2 alkaline	Thermo Scientific	31324
phosphatase-conjugated antibody
Hemagglutinin (HA) peptide, 100 mg	Genscript	custom synthesis
(sequence: YPYDVPDYA; 95% purity)
HEK 293T cells	ATCC	CRL-11268
Household bleach (4% w/v sodium hypochlorite)	various brands are available
Immobilon-P PVDF membrane	Millipore	IPVH07850
MiniPrep plasmid purification kit, PureLink Quick	Invitrogen	K2100-11
MaxiPrep plasmid purification kit, PureLink HiPure	Invitrogen	K2100-07
NaN3	Sigma	S8032
pDISPLAY expression vector	Invitrogen	V660-20
Penicillin/streptomycin (pen/strep), 100X	Invitrogen	15140-122
Phosphate-buffered saline (PBS), sterile 1X	Sigma	D8537
Polyethyleneimine (PEI), linear 25 kDa	Polyscience	23966
Skim milk dry powder	Carnation
Stericup-GP PES vacuum filtration unit,	Millipore	SCGPU05RE
0.22 μm, 500 ml capacity
Trypan blue	Invitrogen	15250061
Trypsin-EDTA, 0.05% (w/v)	Invitrogen	25300-054
Tween-20	Sigma	P7949
Valproic acid	Sigma	P4543
Centramate tangential flow system	Pall
CO2 humidified incubator, standard 6.0 cu. ft.	various brands are available
Electrophoresis and transfer unit	various brands are available
Incubator, 37 °C	various brands are available