This work was supported by NIH R01-HL119813 (to T.J.B.), American Lung Association RG-196051 (to T.J.B.), a CIMED Pilot and Feasibility grant (to T.J.B.), American Heart Association Predoctoral Fellowships 14PRE19970008 (to Z.Y.) and 15PRE22110004 (to D.L.K.).

1. Production of Milligram Quantities of Plasmid DNA for Large-scale Transient Transfection
<ol>
	<li>Clone the protein of interest into a high copy number mammalian expression vector using restriction site cloning, or other appropriate technique.
	<ol>
		<li>For optimal results, use pHLsec 7 vector, which has a built-in C-terminal 6His-tag, a strong promoter Kozak sequence and an optimized secretion signal.</li>
	</ol>
	</li>
	<li>Transform the plasmid onto competent cells.
	<ol>
		<li>Add 20 &#181;l of competent E. coli cells onto 1 &#181;g of plasmid DNA and incubate on ice for 30 min.</li>
		<li>Heat shock cells at 42 &#176;C for 35 sec, then incubate on ice for 2 min.</li>
		<li>Add 300 &#181;l of microbial growth medium (SOC) and incubate at 37 &#176;C for 45 mins, shaking at 220 rpm.</li>
		<li>Plate cells on agar plate with appropriate antibiotic selection.
		<ol>
			<li>Use 100 &#181;g/ml carbenicillin if the plasmid is in the pHLsec vector.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Culture colonies in 250 ml of Luria Broth (LB) Media supplemented with 100 &#181;g/ml antibiotic (carbenicillin) O/N at 37 &#176;C, shaking at 220 rpm.</li>
	<li>Purify DNA from culture using Hi-Speed Plasmid Maxi Kit according to manufacturer&#39;s protocol.
	<ol>
		<li>Elute DNA in buffer EB (10 mM Tris-Cl, pH 8.5), instead of buffer TE.</li>
		<li>Aliquot the purified plasmid at amount needed for transfection and store at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Large-scale Culture and Transient Transfection of 293F Cells
<ol>
	<li>Supplement 1 L 293F media with 10 ml of glutamine and 5 ml Pen/Strep (both 100x). Store at 4 &#176;C. 5 ml Pen/Strep is a sufficient strength in serum-free conditions and the reduced antibiotic concentration improves cell viability during transfection, which improves protein yields.</li>
	<li>Culture 293F cells in 300 ml media in 1 L polycarbonate baffled Erlenmeyer flasks with vented caps at 37 &#176;C with 8% CO2, while shaking in a standard tissue culture incubator.</li>
	<li>Dilute cells to 5 x 105/ml density one day before transfection.</li>
	<li>On the day of transfection, supplement culture medium by adding 10% volume of 2% w/v Cell Boost in 293F media.
	<ol>
		<li>Measure glucose concentration using a glucose monitor according to the manufacturer&#39;s instructions and use supplements as needed to achieve a glucose concentration of 500 mg/dL.</li>
	</ol>
	</li>
	<li>Add kifunensine (1 &#181;g/ml final concentration) at this step to control protein glycosylation.</li>
	<li>Calculate volume of DNA required for 1 &#181;g plasmid per 1 x 106 cells. Under sterile conditions, dilute DNA in 5 ml serum-free medium.</li>
	<li>Calculate volume of transfection reagent required for 1 &#181;g plasmid per 2 &#181;l transfection reagent. Under sterile conditions, dilute transfection reagent (PEI-TMC-25) in 5 ml serum-free medium.</li>
	<li>Add transfection reagent into DNA solution in 1 ml increments, mixing gently. Incubate for 30 min&#160;at RT for reagent-DNA complexes to form. Then add the solution onto the cells in a drop-wise fashion.</li>
	<li>Allow transfected cells to express protein for 72-96 hr. Supplement with ~10% volume Cell Boost Media daily, or as necessary to keep glucose reading 400-600 mg/dl.</li>
</ol>
3. Purification
<ol>
	<li>Decant culture into a centrifuge flask, centrifuge for 20 min at 1,300 x g to pellet cells and then collect the supernatant. If necessary, spin a second time and/or use 0.22 &#181;m filter to clarify supernatant.</li>
	<li>Add 10% volume 10x Ni-NTA binding buffer (1.5 M NaCl, 0.5 M K2HPO4, 0.1 M Tris pH 8.5, 50 mM imidazole).</li>
	<li>Prepare a gravity column by adding 2 ml of Ni-NTA slurry in a column and equilibrating with 10 column volumes (CV) of 1x binding buffer. If possible, do all column steps in a 4 &#176;C room. Alternatively, chill protein and all buffers on ice before column step, and keep protein and collected flow-through on ice. 
	Note: Ni-NTA slurry is 50% resin by volume and the manufacture&#39;s stated binding capacity is 50 mg/ml. Ni-NTA beads can be re-charged for multiple uses</li>
	<li>Flow the supernatant over the resin and collect flow-through. Repeat this step.</li>
	<li>Wash with 10 CV of wash buffer (300 mM NaCl, 50 mM K2HPO4, 20 mM imidazole pH 8).</li>
	<li>Elute the protein in 5 CV of elution buffer (300 mM NaCl, 50 mM K2HPO4, 250 mM imidazole pH 8).</li>
	<li>If deglycosylation is required:
	<ol>
		<li>For a final volume of 0.5 ml, concentrate eluate to 0.43 ml using a centrifugation concentrator. If precipitates form, pellet any debris by centrifugation at 16,000 x g and 4 &#176;C.</li>
		<li>Add 50 &#181;l of 500 mM Na-Citrate pH 5.5.</li>
		<li>Add 20 &#181;l of EndoHf (1 x 106 U/ml). Incubate at RT for 2 hr. 
		Note: The enzyme works optimally at 37 &#176;C, which may cause the concentrated protein to aggregate. Extend the RT incubation, if deglycosylation, assessed by SDS-PAGE or immunoblotting, is incomplete. The enzyme does not have activity at 4 &#176;C.</li>
		<li>To remove EndoHf: Wash Amylose Resin 3x in phosphate buffered saline (PBS) or final storage buffer. Incubate protein with resin for 1 hr at 4 &#176;C. Spin 5 min at 1,000 x g to pellet beads and collect the supernatant.</li>
		<li>Concentrate protein using appropriate molecular weight cutoff centrifugation filter and buffer exchange into storage buffer (150 mM NaCl and 20 mM HEPES pH 7.5).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Meyer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-host+expression+system+for+recombinant+production+of+challenging+proteins.">Multi-host expression system for recombinant production of challenging proteins.</a> PLoS One. 8, e68674 (2013).</li><li>Chang, V. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+structural+genomics:+solving+the+glycosylation+problem.">Glycoprotein structural genomics: solving the glycosylation problem.</a> Structure. 15, 267-273 (2007).</li><li>Rich, J. R., Withers, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+methods+for+the+production+of+homogeneous+human+glycoproteins.">Emerging methods for the production of homogeneous human glycoproteins.</a> Nat Chem Biol. 5, 206-215 (2009).</li><li>Wu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-2+promotes+macrophage+survival+and+lung+disease+after+respiratory+viral+infection.">TREM-2 promotes macrophage survival and lung disease after respiratory viral infection.</a> J Exp Med. 212, 681-697 (2015).</li><li>Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitigated+cytotoxicity+and+tremendously+enhanced+gene+transfection+efficiency+of+PEI+through+facile+one-step+carbamate+modification.">Mitigated cytotoxicity and tremendously enhanced gene transfection efficiency of PEI through facile one-step carbamate modification.</a> Adv Healthc Mater. 2, 1304-1308 (2013).</li><li>Elbein, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosidase+inhibitors:+inhibitors+of+N-linked+oligosaccharide+processing.">Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.</a> FASEB J. 5, 3055-3063 (1991).</li><li>Aricescu, A. R., Lu, W., Jones, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells.">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta Crystallogr D Biol Crystallogr. 62, 1243-1250 (2006).</li><li>Kelker, M. S., Debler, E. W., Wilson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+mouse+triggering+receptor+expressed+on+myeloid+cells+1+(TREM-1)+at+1.76+A.">Crystal structure of mouse triggering receptor expressed on myeloid cells 1 (TREM-1) at 1.76 A.</a> J Mol Biol. 344, 1175-1181 (2004).</li><li>Kober, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+crystallization,+and+preliminary+crystallographic+analysis+of+wild-type+and+mutant+human+TREM-2+ectodomains+linked+to+neurodegenerative+and+inflammatory+diseases.">Preparation, crystallization, and preliminary crystallographic analysis of wild-type and mutant human TREM-2 ectodomains linked to neurodegenerative and inflammatory diseases.</a> Protein Expr Purif. 96, 32-38 (2014).</li><li>Yurtsever, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-cleavage+of+human+CLCA1+protein+by+a+novel+internal+metalloprotease+domain+controls+calcium-activated+chloride+channel+activation.">Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.</a> J Biol Chem. 287, 42138-42149 (2012).</li><li>Sala-Rabanal, M., Yurtsever, Z., Nichols, C. G., Brett, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+CLCA1+modulates+TMEM16A+to+activate+Ca-dependent+chloride+currents+in+human+cells.">Secreted CLCA1 modulates TMEM16A to activate Ca-dependent chloride currents in human cells.</a> Elife. 4, (2015).</li><li>Niesen, F. H., Berglund, H., Vedadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+differential+scanning+fluorimetry+to+detect+ligand+interactions+that+promote+protein+stability.">The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.</a> Nat Protoc. 2, 2212-2221 (2007).</li></ol>

The authors declare that they have no competing financial interests.

HEK 293F cells offer robust production of proteins requiring post-translational modifications. This system allows rapid and scalable expression of natively folded proteins containing disulfides, glycosylation, and phosphorylation that would otherwise be absent using more routine expression tools. In addition, this system can be used for the expression and purification of multi-protein complexes simply by co-transfection of multiple plasmids. Besides TREM2, this system has been extensively used for functional studies with...

Understanding protein structure at an atomic level is key to uncovering the molecular basis of biological pathways and diseases. X-ray protein crystallography is the most widely used/applicable method for determining macromolecular structures. The main challenge of this method is obtaining sufficient amounts of properly folded, pure protein. This becomes an issue particularly when working with secreted mammalian proteins, which undergo specific post-translational modifications.
Bacterially-expressed proteins are the primary source of crystallized proteins deposited in the Protein Data Bank1. Bacterial expression systems are largely preferred because they are fast, inexpensive and typically produce high yields of protein. However, extracellular domains of mammalian proteins expressed in bacteria are often not properly folded, in which case refolding and extensive purification steps are required for obtaining homogeneously folded protein. Additionally, many mammalian proteins require post-translational glycosylation to achieve proper folding2. Although expression and glycosylation in yeast or insect cells can overcome the folding problem, post-translational modifications, including glycosylation, differ significantly from those of mammalian cells3, yielding proteins with incorrect or non-homogeneous modifications.
Mammalian cells express all the required molecular machinery to ensure proper post-translational modifications and folding; however, these expression systems are not typically preferred by most labs, due to limited yields and high costs of reagents and consumables. Polyethyleneimine (PEI), a standard transfection reagent is relatively cheap but imposes considerable cytotoxicity and low transfection efficiency, resulting in increased costs in cell media, DNA, and culturing equipment. Many alternatives to PEI are prohibitively expensive. We address these issues by describing a combination of improved cell culture tools and chemically modified PEI for the quick and relatively inexpensive method for the expression of secreted mammalian proteins, followed by single-step purification. This robust method gives sufficient yields for functional and biochemical studies4, and in some cases, results in protein amenable to crystallization without further purification.
This protocol describes several techniques to maximize expression and yield for secreted mammalian proteins in human embryonic kidney (HEK) 293F cells grown in suspension. Transfection efficiency (and cost), protein production and purification are all greatly enhanced by following this protocol. PEI modified by the addition of carbamates through a single-step ring-opening reaction (PEI-TMC-25, synthesis and properties described in detail in ref 5) greatly improves transfection efficiency, reduces the cytotoxicity from cationic membrane disruption and accordingly reduces experiment costs. Furthermore, cell viability and protein expression are greatly improved with the addition of culture supplements to supply glucose and vitamins. Importantly for the production of glycosylated proteins, treatment with kifunensine, a non-toxic chemical inhibitor of Mannosidase I, produces proteins with defined, immature glycans, which can be removed by the endoglycosidase EndoHf to yield proteins with a single N-acetylglucosamine in place of a full-length N-linked glycan6. Finally, the secretion of proteins into a serum-free, chemically defined medium allows rapid and facile purification for structural and biochemical studies. Single-step nickel-nitrilotriacetic acid (Ni-NTA) resin purification removes the majority of contaminating species in the supernatant and, in some cases, can yield protein of sufficient purity for crystallization.

<table><tbody><tr><td>Culture Flasks</td><td>GeneMate</td><td>F-5909B</td><td></td></tr><tr><td>293 Freestyle Media</td><td>Gibco/Life Technologies</td><td>12338-018</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco/Life Technologies</td><td>35050-061</td><td>Use in place of Glutamine</td></tr><tr><td>Hype 5 transfection reagent</td><td>Oz Biosciences</td><td>HY01500</td><td></td></tr><tr><td>293fectin transfection reagent</td><td>Life Technologies</td><td>12347019</td><td></td></tr><tr><td>PEI transfection reagent</td><td>Sigma-Aldrich</td><td>408727</td><td></td></tr><tr><td>Maxiprep Kit</td><td>Qiagen</td><td>12162</td><td></td></tr><tr><td>Ni-NTA Superflow&amp;nbsp;</td><td>Qiagen</td><td>30430</td><td></td></tr><tr><td>Endo Hf</td><td>NEB</td><td>P0703L</td><td></td></tr><tr><td>Amylose Resin</td><td>NEB</td><td>E8021S</td><td></td></tr><tr><td>Cell Boost R05.2</td><td>HyClone</td><td>SH30584.02</td><td>Cell Culture Supplement</td></tr><tr><td>GlucCell</td><td>CESCO Bioengineering</td><td>DG2032</td><td>Glucose Monitoring System</td></tr><tr><td>Opti-MEM</td><td>Life Technologies</td><td>519850.91</td><td>Serum Free Medium for DNA transfection</td></tr><tr><td>Luria Broth (LB Media)</td><td>Life Technologies</td><td>10855-001</td><td></td></tr><tr><td>GC10 Competent Cells</td><td>Sigma-Aldrich</td><td>G2919</td><td></td></tr></tbody></table>

efficient mammalian cell expression and single-step purification of extracellular glycoproteins for crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

Herein follows the results of this expression system applied to a secreted 13 kDa immunoglobin (Ig) domain from the human protein triggering receptor expressed on myeloid cells 2 (hTREM2, residues 19-132). TREM2 is a type I transmembrane protein containing a single extracellular Ig domain that has two disulfide bonds and two N-linked glycosylation sites. Unlike many other Ig domain proteins8, TREM2 was not amenable to refolding from bacterial inclusion bodies9. Subsequent mutagenesis confirmed N-lin...

Watch this Scientific Journal Video about Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization at JoVE.com

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

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13.7K Views.  Washington University School of Medicine. This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Video: Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Biochemistry

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Crystallization of ABC Transporter Membrane Proteins in Lipidic Bicelles

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an aqueous environment is typically achieved by employing detergents. These detergents disintegrate the lipid bilayer and solubilize the proteins. The intrinsic habitat of membrane proteins within the lipid bilayer poses a challenge in maintaining their stability and uniformity in solution for structural characterization. Bicelles, which comprise a blend of long and short-chain phospholipids and detergents, replicate the natural lipid structure. The utilization of lipid bicelles and detergents serves as a suitable model system for obtaining high-quality diffraction crystals, specifically to determine the high-resolution structure of membrane proteins. Through these synthetic microenvironments, membrane proteins preserve their native conformation and functionality, facilitating the formation of three-dimensional crystals. In this approach, the detergent-solubilized heterodimeric ABCG5/G8 was reintegrated into DMPC/CHAPSO bicelles, supplemented with cholesterol. This setup was employed in the vapor diffusion experimental procedure for protein crystallization.

Author Spotlight: A Bicelle Crystallization Setup for ABC Transporter Membrane Proteins to Advance Drug Development 

This protocol describes a setup for the crystallization of the sterol transporter ABCG5/G8. ABCG5/G8 is reconstituted into bicelles for hanging-drop crystallization. The protocol does not require specialized materials or substrates, making it accessible and easy to adapt in any laboratory for determining the protein structure through X-ray crystallography.

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an ...

Strategies for Expressing and Purifying Solute Carrier Transporters

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

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

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.

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.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

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