High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

Summary

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

Abstract

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Introduction

Producing high yields of appropriately folded and post-translationally modified human proteins for detailed analysis of structure and function remains a significant challenge. A large number of expression systems are available that produce recombinant proteins with native-like function and behavior. Bacterial expression systems, predominantly Escherichia coli strains, represent the most accessible and commonly used tools in the research arena, due to the simplicity of these expression systems, though yeast, plant, insect and mammalian systems are also described^1-4. However, the majority of these systems are incapable of appropriate post-translation....

Protocol

This protocol is sufficient for expression using either HEK293F or HEK293S cells. 

1. Cell Establishment 

1. Culture Inoculation
   Note: All culture manipulation procedures must be carried in a BSL-2 facility and each item brought into the biosafety cabinet must be sterilized by spraying with a 70% ethanol in water solution.
   1. Operate the incubator shaker at 135 rpm, 80% humidity and at 8.0% CO₂ and 37 °C. Turn "on" the UV lamp of the biosafety cabinet at least 1 h prior to working. Prewarm the sealed Medium A and Medium B bottles in the water-bath at 37 °C for 1 hr.
   2. Sterilize 125 ml Erle....

Results

High-level protein expression and purity

This optimized expression system generated a high yield of glycosylated proteins. A typical pattern is shown in the expression of IgG1-Fc (Figure 1). In this case, Day 0 is the transfection day followed by Day 1 (dilution) and subsequent culture days up to Day 5. Protein expression is analyzed using the soluble expression fraction in the crude medium. A very small amount .......

Discussion

This protocol illustrates protein expression via the transient transfection of HEK293F or S cells. The optimal transfection conditions established in the Barb and Moremen labs employ a critical combination of cell density and reagent concentrations to achieve high efficiency transfection. Critical considerations when implementing this protocol include: maintaining a stable culture prior to transfection (with consistent culture doubling times); transfection of actively growing cells (achieved by diluting cells to 1 ×.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biosafety cabinet	NuAire, Inc.	CellGard ES NU-S475-400	Class II, Type A2 Biological Safety Cabinet
Incubation shaker	INFORS HT		Multitron Cell
Medium A: FreeStyle Expression Medium	Life Technologies	12338-018
Medium B: ExCell 293 Serum-Free Medium	SIGMA	14571C
125 Erlenmeyer Flask with Vented Cap	Corning Incorporated/Life Sciences	431143
250 Erlenmeyer Flask with Vented Cap	Corning Incorporated/Life Sciences	431144
FreeStyle HEK 293F Cells	Life Technologies	R790-07
1 ml Disposable serological pipette	Fisher Scietific	13-676-10B
10 ml Disposable serological pipette	Fisher Scietific	13-676-10J
25 ml Disposable serological pipette	Fisher Scietific	13-676-10K
Pipettor (Pipet-Aid XP)	Drummond Scientific	161263
Trypan Blue Solution	Thermo Scientific	SV30084.01
Counting slides	Bio-Rad	145-0011
TC20 Automated Cell Counter	Bio-Rad	145-0102
Polyethylenimine (PEI)	Polysciences Inc.	23966	Prepare stock solution at a concentration of 1 mg/ml in a buffer containing 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 150 mM NaCl (pH 7.5). Dissolve PEI completely; sterilize through 0.22 μM syringe filter and store at -20 °C.
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	EMD Chemicals Inc.	7365-45-9
25 mm Syringe Filter, 0.22 μM	Fisher Scietific	09-719A
Trisaminomethane (Tris base)	Fisher Scietific	BP152-1
XL1-Blue	Stratagene	222249
Trypton	Fisher Scietific	BP1421-2
Yeast extract	Fluka Analytical	92144
Sodium chloride	BDH chemicals	BDH8014
Plasmid Purification Kit	QIAGEN	12145
Valproic (VPA)	SIGMA	P4543	Prepare stock solution of 220 mM  in water, sterilize by passage through a sterile 0.22 mm filter and store at -20 °C
Centrifuge	Thermo Scientific	EW-17707-65
Protein A-Sepharose column	SIGMA	P9424
Ni-NTA superflow	QIAGEN	30430
3-(N-morpholino)propanesulfonic acid (MOPS)	Fisher Scietific	BP308-500
Glycine	Fisher Scietific	BP381-500
10 kDa molecular weight cut-off Amicon® Ultra centrifugal filters	Millipore	UFC901096
Sodium dodecyl sulfate	ALDRICH	L3771
Beta-mercaptoethanol	ALDRICH	M6250
Glycerol	SIGMA	G5516
Precision Plus Protein All Blue Standards	Bio-Rad	161-0373
Acetic Acid, Glacial	Fisher Scietific	64-19-7
coomassie brilliant blue	Bio-Rad	161-0406
MALDI-TOFMS Voyager-DE PRO	Applied Biosystems
15N labeled L-Tyrosine	ALDRICH	332151
15N labeled L-Lysine	ALDRICH	592900
Unlabeled L-Phenylalanine	SIGMA-ALDRICH	P2126
13C6-Glucose	ALDRICH	389374
2-deoxy-2-fluoro-l-fucose	SANTA CRUZ Biotechnology	sc-283123