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Glycoproteins mediate multiple, diverse and critical cellular functions, that are desirable to explore by structural analysis. However, structure determination of these molecules has been hindered by difficulties expressing milligram quantities of stable, homogeneous protein and in determining, which modifications will yield samples amenable to structural studies. We describe a platform proven effective for rapidly screening expression and crystallization of challenging glycoprotein targets produced in mammalian cells. Here, multiple glycoprotein constructs are produced in parallel by transient expression of adherent human embryonic kidney (HEK) 293T cells and subsequently screened in small quantities for crystallization by microfluidic free interface diffusion. As a result, recombinant proteins are produced and processed in a native, mammalian environment and crystallization screening can be accomplished with as little as 65 μg of protein. Moreover, large numbers of constructs can be screened for expression and crystallization and scaled up for structural studies in a matter of five weeks.
Glycoproteins are thought to make up about 50% of all human and human-pathogen proteins, but comprise only 3% of structures deposited in the Protein Data Bank (PDB). Structural biology of glycoproteins is hindered by challenges in achieving high-level expression of soluble, stable and homogenous protein capable of forming well-diffracting crystals. Although attachment and processing of glycans is often a prerequisite for correct protein folding and stability, the chemical and conformational heterogeneity of complex-type glycans displayed on proteins produced in mammalian cells generally inhibits formation of a well-ordered crystal lattice1. Multiple rounds of iterative construct re-design or site-directed mutagenesis of N- or O-linked glycosylation sequons, coupled with enzymatic deglycosylation, are usually necessary to identify a protein construct suitable for crystallization. Glycoprotein expression efforts often use Chinese hamster ovary (CHO) cells2, yeast3 and insect cell-based systems4-7. However, these systems are not ideal for rapid construct screening as initial setup of each stable cell line can be time-consuming. Moreover, processing of glycans in yeast, insect and Drosophila cell lines differs from that which occurs in human glycan biosynthetic pathways, possibly influencing protein folding, stability, activity, secretion and immunogenicity8-11. Recent developments using transient transfection protocols of adherent human embryonic kidney (HEK) 293T cells have shown greater promise for higher throughput of mammalian-expressed glycoproteins12,13.
We describe a procedure for screening crystallization using transient expression in adherent HEK293T cells, one-step affinity purification, and microfluidic free interface diffusion. This platform allows cloning, expression and crystallization to be performed in approximately 5 weeks, significantly faster than current protocols. This protocol is now in standard use for all mammalian-expressed glycoproteins in our laboratory14 and by a number of other groups12,13. Its utility is proven by the successful structural determination of a challenging 320 kDa Zaire ebolavirus glycoprotein-antibody complex14.
The main considerations in the selection of a vector for expression of mammalian glycoproteins are:
While many commercial vectors are available for expression of human proteins, we have found the pDISPLAY expression system to be of particular utility. The pDISPLAY plasmid has a vector-encoded, C-terminal transmembrane domain and an Ig κ chain leader sequence which targets the protein through the ER and Golgi networks for display on the cell surface. We usually insert a stop codon prior to the vector-encoded transmembrane anchor to allow soluble protein to be released into the cultured media. A strong human cytomegalovirus (CMV) protomer ensures over-expression of the protein of interest, and the presence of a hemagglutinin (HA) tag allows the secreted protein to be selectively purified by immunoaffinity chromatography and detected by Western blot.
Adherent HEK293T cells (Figure 2) are easy to maintain, able to express and properly process recombinant human proteins12,13, and highly suitable for use in small- to medium-sized laboratories. HEK293T cells are available from major cell banks such as ATCC, and are easily cultured using Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine (1× GlutaMAX) and 5% (v/v) fetal bovine serum (FBS) in a 37°C incubator with 5% CO2. Test expressions are performed in 6-well (9.5 cm2 surface area) or in 24-well (1.9 cm2 surface area) cell culture plates. High-level expression in small volumes is best obtained when cells are transfected at 70% confluency and when a commercial reagent such as FuGENE HD (Roche) or GeneJuice (EMD Biosciences) is used for transfection. The detection and analysis of dilute amounts of protein secreted into conditioned media is performed by Western blot analysis using antibodies directed against both linear- and conformational epitopes. The use of an antibody against a linear epitope allows detection of total protein expressed, regardless of quality, while use of an antibody against a conformational epitope (if available) distinguishes properly folded from misfolded protein.
A variety of containers, such as T225 cm2 flasks, roller bottles (850 cm2) or CellSTACKS (636 cm2/layer) can be used for 293T culture and expression. We favour the Corning 10-layer CellSTACK system for larger scale expression and 2- or 5-layer CellSTACKS for smaller scale exploratory studies (Figure 2). CellSTACKS provide equivalent or larger surface area for cell attachment and growth than other vessels, yet package cell culture space effectively, thus requiring less incubator space and labour. Although disposable CellSTACKS are more costly than roller bottles, they can be made more cost-effective by washing and recycling the vessel.
While FuGENE HD is often used in our laboratory for test transfections, the cost of this reagent is prohibitively expensive for use in large-scale expression. We have found that the use of traditional inexpensive transfection reagents, such as calcium phosphate (reagent of choice in our laboratory) or branched PEI12,15,16, provides excellent transfection efficiencies and cost savings.
In our laboratory, glycoproteins are secreted into litre-scale volumes of conditioned media, and are efficiently purified via a two-step procedure. First, large volumes of media are rapidly concentrated to more manageable amounts using a Centramate tangential flow filtration system. This is followed by capture of the recombinant protein by affinity chromatography on an anti-HA column with high affinity and capacity. Bound proteins are gently eluted from anti-HA columns by competition with a synthetic HA peptide. Afterwards, the anti-HA affinity matrix can be regenerated multiple times without loss of performance. Alternatively, dilute concentrations of secreted protein may be loaded directly onto the affinity column without prior concentration, although this process is slower, or proteins may be fractionated by ammonium sulfate precipitation and reconstituted prior to affinity capture. Many other affinity purification tags such as biotin, glutathione S-transferase (GST), myc-epitope, FLAG, and poly-histidine have also been developed, however we prefer the hemagglutinin tags because of the ease of the antibody-based purification and low cross-reactivity with host cell proteins. In our experience, low-yield proteins purified by metal affinity chromatography retain a number of host cell contaminants and require additional purification steps before crystallization.
N-linked glycans attached to proteins expressed in mammalian cells are usually complex, chemically and conformationally heterogeneous in nature, and frequently detrimental to formation of well-ordered crystal lattices. Several strategies have been utilized to overcome the “glycosylation problem”. Two popular methods involve using either a N-acetylglycosaminyltransferase I (GnT I)-deficient HEK293S cell line17 or addition of glycan anabolic inhibitors such as swainsonine or kifunensine13 to cell culture media. Both strategies yield more homogeneous, high mannose-type oligosaccharide chains, which can be removed by EndoH deglycosylation13.
An alternate approach that we favour, involves production of native glycoproteins without inhibitors and enzymatically deglycosylate the resulting protein under native conditions using peptide N-glycosidase F (PNGaseF), followed by Endoglycosidase (Endo)F1, EndoF2 or EndoF3. Each of these enzymes has a unique glycan substrate preference (Figure 3). Of these, PNGaseF has the broadest substrate specificities and cleaves between the asparagine side chain and the innermost N-acetylglucosamine (GlcNAc), thus removing the entire glycan moiety. Occasionally, some glycan attachment sites are buried or masked by the local protein structure and PNGaseF is unable to gain access. However, the EndoF series of glycosidases, which cleave between the two GlcNAc oligosaccharides in the chitobiose core, are less sensitive to protein conformation, and may trim glycans where PNGaseF has failed. In addition, in some cases, removal of terminal sialic acids by neuraminadase, may be beneficial prior to further deglycosylation with EndoF or PNGaseF.
The use of robotic liquid handling systems has significantly reduced time and protein quantity required for crystallization screening. Indeed, current robots are able to accurately dispense ~100 nL of protein or reagent per drop. A recent, additional improvement in protein crystallization is the development of microfluidic circuits to dispense even smaller volumes of protein and reagents for crystallization. For example, the Fluidigm Topaz system allows screening of 96 different crystallization conditions with a mere 1.5 μL of protein, which can be critical for crystallization targets that are difficult to produce. Indeed, an increasing number of mammalian protein structures, including those of glycosylated proteins, have been determined from initial crystallization hits obtained from microfluidic-based diffusion18-25. The system is quite expensive, but in our hands, has been a key determinant in the feasibility of structural analysis of some difficult targets. The Topaz microfluidic screening chips (Figure 4) contain a series of interconnected miniaturized channels and elastomeric rubber valves that control the interface of protein sample and precipitant. Initially, diffusion of protein and precipitant sets a concentration gradient to allow the sampling of a wide area of crystallization space. Subsequently, vapour permeability of the reaction chamber allows dehydration to further concentrate the sample, yielding results in 7 days. Quality hits can then be translated into standard 0.5-2.0 μL size hanging drop, vapour diffusion experiments.
|Glycosidase||Units of glycosidase||Protein (μg)||Buffer||Final volume (μL)|
|PNGaseF||500||40||8 μL 10× PBS pH 7.4||80|
|EndoF1||0.04||40||8 μL 1M sodium citrate pH 5.5||80|
|EndoF2||0.01||40||8 μL 1M sodium acetate pH 4.5||80|
|EndoF3||0.01||40||8 μL 1M sodium citrate pH 5.5||80|
The following is a summary of the Topaz microfluidic crystallization process; consult the manufacturer's instructions for more detailed information.
Exhaustive protocols on tissue culture and expression are available in sources such as Cold Spring Harbor Protocols. In Table 1, we present some of the more common problems and their solutions.
The expression and crystallization platform described here was instrumental in determination of the structure of the trimeric, prefusion Zaire ebolavirus glycoprotein (EBOV GP)14. Crystal structures of viral glycoproteins are difficult to achieve given the challenges to production, purification, crystallization and diffraction inherent in the heavily glycosylated, oligomeric, metastable, and flexible nature of these proteins. In the case of EBOV GP, the strategy described here allowed rapid expression screening for over 100 constructs containing various truncations and multiple combinations of mutations to N-x-S/T glycosylation sequons. Moreover, the HEK293T cell productions could be scaled up to quantities which allowed growth and screening of 50,000 crystals. It is anticipated that this platform will be a useful tool not just for glycosylated proteins, but also for mammalian proteins in general.
Constructs selected for scale-up should be characterized by a single strong band at the proper molecular weight on both linear anti-HA and conformation-dependent antibody immunoblots, indicating adequate expression level, monodispersity and proper folding (Figure 5a, for one example). In our laboratory, large-scale expression in HEK293T cells routinely results in the production of ~2-10 mg of protein/L of media. It is expected that glycans produced in HEK293T cells can be efficiently removed by either PNGaseF or the EndoF series of glycosidases (Figure 5b). However, deglycosylation is often incomplete due to poor steric access of the attachment site by the local conformation of the protein. In the case of EBOV GP, resistant glycan sites were removed by site-directed mutagenesis of the N-x-S/T glycosylation sequons14.
Fluidigm microfluidic free interface diffusion crystallization uses only 6 μL of protein (10 mg/mL) to screen 384 sparse matrix conditions and yields results within 7 days. Protein crystals usually appear closer to the right-hand side of the chamber, which is the region of highest protein concentration. However, the concentration gradient formed in the horizontal axis of the chamber may sometimes permit crystallization in the middle of the chamber. Crystals closest to or contained within the precipitant inlet side (far left) are usually salt. Crystallization hits can be scored into four classes (I-IV; Figure 6). Class I crystals appear perfect, with sharp edges and thickness in three dimensions. Class II crystals are similar to class I, but have minor defects, such as irregular, jagged or rounded edges or satellites. Class III crystals are small needle or crystalline clusters, while class IV crystals are showers of microcrystals or spherulites. In the case of EBOV GP and other projects in our group, the success of translation is highly dependent on the quality of the crystal hit and number of unique hits obtained. Class I and II crystal hits have high translation percentages and can often be translated directly into similar conditions of the crystallization screen. Translation of class III and IV crystals tends to be difficult and likely requires additional optimization of pH, salts, PEG or additives using a 1.96 chip to yield more crystalline class I and II-type crystals prior to translation.
The authors would like to thank Dr. Robyn Stanfield at The Scripps Research Institute for assistance with the Fluidigm microfluidic system and members of the Ollmann Saphire laboratory for advice and help. This work is supported by NIH NIAID operating grants (AI053423 and AI067927) and a Career Award by the Burroughs Wellcome Fund to E.O.S. and a fellowship to J.E.L. from the Canadian Institutes of Health Research. This is manuscript #XXXXX from The Scripps Research Institute.