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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Production of Milligram Quantities of Plasmid DNA for Large-scale Transient Transfection

1. Clone the protein of interest into a high copy number mammalian expression vector using restriction site cloning, or other appropriate technique.
   1. 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.
2. Transform the plasmid onto competent cells.
   1. Add 20 µl of competent E. coli cells onto 1 µg of plasmid DNA and incubate on ice for 30 min.
   2. Heat shock cells at 42 °C for 35 sec, then incubate on ice for 2 min.
   3. Add 300 µl of microbial growth medium (SOC) and incubate at 37 °C for 45 mins, shaking at 220 rpm.
   4. Plate cells on agar plate with appropriate antibiotic selection.
      1. Use 100 µg/ml carbenicillin if the plasmid is in the pHLsec vector.
3. Culture colonies in 250 ml of Luria Broth (LB) Media supplemented with 100 µg/ml antibiotic (carbenicillin) O/N at 37 °C, shaking at 220 rpm.
4. Purify DNA from culture using Hi-Speed Plasmid Maxi Kit according to manufacturer's protocol.
   1. Elute DNA in buffer EB (10 mM Tris-Cl, pH 8.5), instead of buffer TE.
   2. Aliquot the purified plasmid at amount needed for transfection and store at -20 °C.

2. Large-scale Culture and Transient Transfection of 293F Cells

1. Supplement 1 L 293F media with 10 ml of glutamine and 5 ml Pen/Strep (both 100x). Store at 4 °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.
2. Culture 293F cells in 300 ml media in 1 L polycarbonate baffled Erlenmeyer flasks with vented caps at 37 °C with 8% CO₂, while shaking in a standard tissue culture incubator.
3. Dilute cells to 5 x 10⁵/ml density one day before transfection.
4. On the day of transfection, supplement culture medium by adding 10% volume of 2% w/v Cell Boost in 293F media.
   1. Measure glucose concentration using a glucose monitor according to the manufacturer's instructions and use supplements as needed to achieve a glucose concentration of 500 mg/dL.
5. Add kifunensine (1 µg/ml final concentration) at this step to control protein glycosylation.
6. Calculate volume of DNA required for 1 µg plasmid per 1 x 10⁶ cells. Under sterile conditions, dilute DNA in 5 ml serum-free medium.
7. Calculate volume of transfection reagent required for 1 µg plasmid per 2 µl transfection reagent. Under sterile conditions, dilute transfection reagent (PEI-TMC-25) in 5 ml serum-free medium.
8. Add transfection reagent into DNA solution in 1 ml increments, mixing gently. Incubate for 30 min at RT for reagent-DNA complexes to form. Then add the solution onto the cells in a drop-wise fashion.
9. 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.

3. Purification

1. 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 µm filter to clarify supernatant.
2. 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).
3. 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 °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's stated binding capacity is 50 mg/ml. Ni-NTA beads can be re-charged for multiple uses
4. Flow the supernatant over the resin and collect flow-through. Repeat this step.
5. Wash with 10 CV of wash buffer (300 mM NaCl, 50 mM K₂HPO₄, 20 mM imidazole pH 8).
6. Elute the protein in 5 CV of elution buffer (300 mM NaCl, 50 mM K₂HPO₄, 250 mM imidazole pH 8).
7. If deglycosylation is required:
   1. 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 °C.
   2. Add 50 µl of 500 mM Na-Citrate pH 5.5.
   3. Add 20 µl of EndoHf (1 x 10⁶ U/ml). Incubate at RT for 2 hr.
      Note: The enzyme works optimally at 37 °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 °C.
   4. 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 °C. Spin 5 min at 1,000 x g to pellet beads and collect the supernatant.
   5. 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).

Results

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...

Discussion

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...

Materials

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