Thermo Fisher Scientific Expi293 User Manual
Expi293™ Expression System: Structural
Biology and Inducible Expression Modules
USER GUIDE
For scalable transfection of Expi293F™ GnTI- and Expi293F™ Inducible cell lines in
a chemically defined, serum-free medium, using the ExpiFectamine™ 293
Transfection Kit.
For scalable metabolic protein labeling using Expi293F™ cell lines in a chemically
defined, serum-free, methionine-free medium using a methionine-deficient
ExpiFectamine™ 293 Transfection Kit.
Catalog Numbers A39250, A39250CN, A39251, A14527CN, A39252, A41276CN, A41249, A14524,
A14525, A14526, A39249
Publication Number MAN0007814
Revision B.0
For Research Use Only. Not for use in diagnostic procedures.
Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, CA 92008
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The information in this guide is subject to change without notice.
DISCLAIMER: T
O THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0007814
Revision Date Description
B.0 29
A.0 14 April 2019 Update of user guide to bring it up to current standards and styles.
2.0 11 June 2018 Format conversion. Addition of application notes.
1.0 June 2013 Introduction of new format
— 2012 New document
May 2020
Update to reflect inducible expression and structural biology kit
configurations.
Addition of GNTI cells, Inducible cells, and Inducible GNTI cells.
Updating of protocols to incorporate the new cell lines added.
Important Licensing Information: This pr
oduct may be covered by one or more Limited Use Label Licenses. By use of this product,
you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 Product information .................................................. 6
Product description ............................................................. 6
Contents and storage ............................................................ 7
Components of the Expi293™ Expression System: structural biology and
inducible expression modules ................................................. 10
Expi293F™ Cells ........................................................... 10
Expi293F™ GnTI- Cells ..................................................... 10
Expi293F™ Inducible Cells .................................................. 10
Expi293F™ Inducible GnTI- Cells ............................................. 10
Expi293™ Expression Medium ............................................... 11
Expi293™ Met (-) Expression Medium ........................................ 11
ExpiFectamine™ 293 Reagent ............................................... 12
ExpiFectamine™ 293 Transfection Enhancer 1 ................................. 12
ExpiFectamine™ 293 Transfection Enhancer 2 ................................. 12
ExpiFectamine™ 293 Met (-) Transfection Enhancer 2 .......................... 12
Opti-Plex™ Complexation Buer ............................................ 13
pRABBIT IgG IRES-EmGFP Positive Control Vector ............................ 13
pcDNA™5/TO Mammalian Expression Vector .................................. 13
PNGase F Glycan Cleavage Kit .............................................. 13
■
Expi293
CHAPTER 2 Methods ............................................................. 14
Procedural guidelines for Expi293F™ cell culture ................................... 14
General cell handling ....................................................... 14
Guidelines for thawing and storing cells ...................................... 14
Guidelines for cell maintenance and subculturing .............................. 14
Guidelines for inducible cells ................................................ 15
Guidelines for media ....................................................... 16
Thaw and establish Expi293F™ cell lines .......................................... 16
Required materials ......................................................... 16
Thaw Expi293F™ cells ...................................................... 16
Subculture Expi293F™ cell lines ................................................. 17
Required materials ......................................................... 17
Subculture Expi293F™ cells ................................................. 18
Cryopreserve Expi293F™ cells ............................................... 19
™
Expression System User Guide
3
Contents
Transfect Expi293F™ cell lines using ExpiFectamine™ 293 Transfection Kit ............. 19
Required materials ......................................................... 20
Guidelines for transfection .................................................. 20
Day −2: Subculture cells ................................................... 21
Day −1: Seed cells ........................................................ 21
Day 0: Transfect cells ...................................................... 21
Day +1: Add Enhancers .................................................... 22
Day +5: Harvest protein .................................................... 22
Transfect Expi293F™ cell lines using ExpiFectamine™ 293 Met (-) Transfection Kit
for metabolic protein labeling .................................................. 22
Required materials ......................................................... 23
Guidelines for transfection .................................................. 23
Day −2: Subculture cells ................................................... 23
Day −1: Seed cells ........................................................ 24
Day 0: Perform metabolic labeling ........................................... 24
Day 0: Transfect cells ...................................................... 25
Day +1: Add Enhancers .................................................... 25
Day +5: Harvest protein .................................................... 26
■
APPENDIX A Additional guidelines .............................................. 27
Guidelines to optimize protein expression ......................................... 27
Equipment .................................................................... 27
Cells ......................................................................... 28
Plasmid DNA complexation ..................................................... 28
Harvest ....................................................................... 28
Cell culture supernatant clarification .............................................. 29
■
APPENDIX B Positive control for transfection and expression ................ 30
pRABBIT IgG IRES-EmGFP Positive Control Vector ................................ 30
Transfection and expression ..................................................... 30
■
APPENDIX C Scaling up transfections .......................................... 31
Scale up transfections .......................................................... 31
■
APPENDIX D Ordering information .............................................. 33
Additional products ............................................................ 33
Shaker flasks for suspension culture .............................................. 34
Plasmid purification products .................................................... 34
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Expi293™ Expression System User Guide
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APPENDIX E Safety ............................................................... 35
Chemical safety ................................................................ 36
Biological hazard safety ......................................................... 37
■
APPENDIX F Documentation and support ...................................... 38
Customer and technical support ................................................. 38
Limited product warranty ........................................................ 38
Contents
Expi293™ Expression System User Guide
5
1
IMPORTANT! Before using this product, read and understand the information in the
“Safety” appendix in this document.
Product description
The Gibco™ Expi293™ Expression System is a high-yield transient expression system
based on suspension-adapted Human Embryonic Kidney (HEK) cells. As part of the
structural biology and inducible expression modules, we oer 3 System Starter Kits
for structural biology or inducible expression and additional reagents to allow
metabolic labelling of proteins. Each Expi293™ Expression System Starter Kit
provides cells, culture medium, and reagents to express a total of 1 liter production
volume.
The structural biology and inducible expression modules of the Expi293™ Expression
System includes four Expi293F™ cell lines that can be used to express your protein of
interest.
•
Use Expi293F™ cells for metabolic protein labeling.
•
Use Expi293F™ GnTI- Cells for expression of proteins with a uniform
glycosylation pattern.
•
Use Expi293F™ Inducible Cells for regulated expression of proteins through
tetracycline mediated induction.
•
Use Expi293F™ Inducible GnTI- Cells for regulated expression of proteins with a
uniform glycosylation pattern.
Product information
Each of these cell lines display the same growth kinetics, follow the same
transfection protocols, and can be used in metabolic protein labeling studies.
For metabolic (methionine) protein labeling, Expi293™ Met (-) Expression Medium and
the ExpiFectamine™ 293 Met (-) Transfection Kit should be used to metabolically label
proteins of interest with L-Methionine (Methyl-13C) or L-Selenomethionine.
Expi293™ Met (-) Expression Medium and the ExpiFectamine™ 293 Met (-)
Transfection Kit can be ordered separately or together as part of the Expi293™ Met (-)
Protein Labeling Kit. L-Methionine (Methyl-13C) and L-Selenomethionine can be
ordered separately.
For further instruction on metabolically labeling proteins using the Expi293
Expression System, see “Transfect Expi293F™ cell lines using ExpiFectamine™ 293
Met (-) Transfection Kit for metabolic protein labeling” on page 22.
6
Expi293™ Expression System User Guide
™
Chapter 1
Contents and storage
Table 1 Expi293™ GnTI- Expression System Kit (Cat. No. A39250 or A39250CN)
Contents Amount Cat. No. Storage
Product information
Contents and storage
1
Expi293F™ GnTI- Cells
[1]
(1 × 107 cells/mL) 1 mL A39240 Liquid nitrogen
Expi293™ Expression Medium 1 L A1435101
ExpiFectamine™ 293 Transfection Kit:
•
ExpiFectamine™ 293 Reagent
•
ExpiFectamine™ 293 Transfection Enhancer 1
•
ExpiFectamine™ 293 Transfection Enhancer 2
1 kit for 1 L of
culture
A14524
2°C to 8°C
Protect from light
Opti-Plex™ Complexation Buer 100 mL A4096801
pRABBIT IgG IRES-EmGFP Positive Control Vector (at
1 mg/mL in TE
[3]
Buer, pH 8.0)
150 µg A39243 −20°C
PNGase F Glycan Cleavage Kit 1 Kit A39245 −20°C
[1]
Cells are cryopreserved in 90% Expi293™ Expression Medium and 10% DMSO.
[2]
Store the frozen cells in liquid nitrogen immediately upon receipt and until ready to use. Do not store the cells at −80°C.
[3]
TE buffer, pH 8.0: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
Table 2 Expi293™ Inducible Expression System Kit (Cat. No. A39251 or A14527CN)
Expi293F™ Inducible Cells
Contents
[1]
(1 × 107 cells/mL) 1 mL A39241 Liquid nitrogen
Amount Cat. No. Storage
[2]
[2]
Expi293™ Expression Medium 1 L A1435101
ExpiFectamine™ 293 Transfection Kit:
•
ExpiFectamine™ 293 Reagent
•
ExpiFectamine™ 293 Transfection Enhancer 1
•
ExpiFectamine™ 293 Transfection Enhancer 2
1 kit for 1 L of
culture
A14524
Opti-Plex™ Complexation Buer 100 mL A4096801
pRABBIT IgG IRES-EmGFP Positive Control Vector (at
1 mg/mL in TE
[3]
Buer, pH 8.0)
150 µg A39243
pcDNA™5/TO Mammalian Expression Vector 20 µg V103320
Tetracycline Hydrochloride
[1]
Cells are cryopreserved in 90% Expi293™ Expression Medium and 10% DMSO.
[2]
Store the frozen cells in liquid nitrogen immediately upon receipt and until ready to use. Do not store the cells at −80°C.
[3]
TE buffer, pH 8.0: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
[4]
Not included in Cat. No. A41275.
[4]
500 mg A39246 Protect from light
2°C to 8°C
Protect from light
−20°C
and moisture
Room temperature
Expi293™ Expression System User Guide
7
Chapter 1 Product information
1
Contents and storage
Table 3 Expi293™ Inducible GnTI- Expression System Kit (Cat. No. A39252 or A41276CN)
Contents Amount Cat. No. Storage
Expi293F™ Inducible GnTI- Cells
[1]
(1 × 107 cells/mL) 1 mL A39242 Liquid nitrogen
Expi293™ Expression Medium 1 L A1435101
ExpiFectamine™ 293 Transfection Kit:
•
ExpiFectamine™ 293 Reagent
•
ExpiFectamine™ 293 Transfection Enhancer 1
•
ExpiFectamine™ 293 Transfection Enhancer 2
1 kit for 1 L of
culture
A14524
2°C to 8°C
Protect from light
Opti-Plex™ Complexation Buer 100 mL A4096801
pRABBIT IgG IRES-EmGFP Positive Control Vector (at
1 mg/mL in TE
[3]
Buer, pH 8.0)
150 µg A39243
−20°C
pcDNA™5/TO Mammalian Expression Vector 20 µg V103320
Tetracycline Hydrochloride
[4]
500 mg A39246 Protect from light
and moisture
Room temperature
PNGase F Glycan Cleavage Kit 1 kit A39245 −20°C
[1]
Cells are cryopreserved in 90% Expi293™ Expression Medium and 10% DMSO.
[2]
Store the frozen cells in liquid nitrogen immediately upon receipt and until ready to use. Do not store the cells at −80°C.
[3]
TE buffer, pH 8.0: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
[4]
Not included in Cat. No. A41276.
[2]
Table 4 Expi293™ Met (-) Protein Labeling Kit (Cat. No. A41249)
Contents
ExpiFectamine™ 293 Met (-) Transfection Kit
•
ExpiFectamine™ 293 Reagent
•
ExpiFectamine™ 293 Transfection Enhancer 1
•
ExpiFectamine™ 293 Met (-) Transfection Enhancer 2
•
Opti-Plex™ Complexation Buer
Amount Cat. No. Storage
1 kit for 1 L
A39249
culture
Expi293™ Met (-) Expression Medium 1 L A4096701
2°C to 8°C
Protect from light.
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Expi293™ Expression System User Guide
Chapter 1 Product information
Table 5 ExpiFectamine™ 293 Transfection Kit (Cat. No. A14524, A14525, and A14526)
Contents Amount Cat. No. Storage
ExpiFectamine™ 293 Transfection Kit:
Contents and storage
1
•
ExpiFectamine™ 293 Reagent
•
ExpiFectamine™ 293 Transfection Enhancer 1
•
ExpiFectamine™ 293 Transfection Enhancer 2
•
1 kit for 1 L
of culture
•
1 kit for 10 L
of culture
•
1 kit for 50 L
of culture
Table 6 ExpiFectamine™ 293 Met (-) Transfection Kit (Cat. No. A39249)
Contents Amount Cat. No. Storage
ExpiFectamine™ 293 Met (-) Transfection Kit
•
ExpiFectamine™ 293 Reagent
•
ExpiFectamine™ 293 Transfection Enhancer 1
•
ExpiFectamine™ 293 Met (-) Transfection Enhancer 2
•
Opti-Plex™ Complexation Buer
1 kit for 1 L
culture
•
A14524
•
A14525
•
A14526
A39249
2°C to 8°C
Protect from light.
2°C to 8°C
Protect from light.
Expi293™ Expression System User Guide
9
Chapter 1
1
Components of the Expi293™ Expression System: structural biology and inducible expression modules
Product information
Components of the Expi293™ Expression System: structural
biology and inducible expression modules
The Expi293™ Expression System is based on high density culture of Expi293F™ Cells
in Expi293™ Expression Medium. Transient expression is by the cationic lipid-based
ExpiFectamine™ 293 Reagent with specialized transfection enhancers. The
components work together to generate 2- to 10-fold higher protein yields than
previous generation transient expression systems.
Expi293F™ Cells
Expi293F™ Cells are human cells derived from the 293F cell line, and are a core
component of the Expi293™ Expression System. They are maintained in suspension
culture and will grow to high density in Expi293™ Expression Medium. Expi293F
Cells are highly transfectable and generate superior protein yields compared to
standard 293 cell lines in transient protein expression.
Expi293F™ GnTI- Cells
™
Expi293F™ GnTI- Cells are derived from Expi293F™ Cells and have been engineered
to lack N-acetylglucosaminyl-transferase I (GnTI) enzyme activity leading to the
production of glycoproteins with a uniform (GlcNAc)2Man5 glycopattern.
Expi293F™ Inducible Cells
Expi293F™ Inducible Cells are derived from Expi293F™ Cells and stably express high
levels of the tetracycline repressor protein (TetR) from the pcDNA™6/TR plasmid.
When used with compatible inducible vectors (e.g. pcDNA™5/TO Mammalian
Expression Vector), the gene of interest is repressed with very low levels of basal
expression until induction with tetracycline. Expression can be modulated by addition
of dierent amounts of tetracycline. For best results, Expi293F™ Inducible Cells
should be maintained under selective pressure of blasticidin. For further details, see
“Guidelines for inducible cells” on page 15.
Expi293F™ Inducible GnTI- Cells
Expi293F™ Inducible GnTI- Cells are derived from Expi293F™ GnTI- Cells and stably
express high levels of the tetracycline repressor protein (TetR) from the pcDNA™6/TR
plasmid in combination with knockout of the GnTI gene. When used with compatible
inducible vectors (e.g. pcDNA™5/TO Mammalian Expression Vector), the gene of
interest is repressed with very low levels of basal expression until induction with
tetracycline. Expression can be modulated by addition of dierent amounts of
tetracycline. Resultant glycan patterns of the expressed glycoprotein are consistent
with those expressed by Expi293F™ GnTI- Cells. For best results, Expi293F
Inducible GnTI- Cells should be maintained under selective pressure of blasticidin.
For further details, see “Guidelines for inducible cells” on page 15.
™
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Expi293™ Expression System User Guide
Components of the Expi293™ Expression System: structural biology and inducible expression modules
Expi293™ Expression Medium
Expi293™ Expression Medium is a chemically defined, serum-free, protein-free,
animal origin-free medium for growth and transfection of suspension-adapted human
embryonic kidney (HEK) 293 cells. It is specifically designed to support high density
culture of Expi293F™ cell lines as part of the Expi293™ Expression System for
scalable transient protein expression. Expi293™ Expression Medium is formulated
with GlutaMAX™ Supplement. It is ready to use and no supplementation is required.
The chemically defined formulation results in high lot-to-lot reproducibility and
reliability. Expi293™ Expression Medium contains no human or animal-origin
components. The medium is not recommended for adherent cell cultures.
Expi293™ Expression Medium exhibits the following features:
•
Supports growth of suspension Expi293F™ cultures to densities over
15 × 106 cells/mL.
•
Transfection compatible medium enables transfection eciencies of
approximately 80% using ExpiFectamine™ 293 Transfection Reagent
•
Enables sustained, high-level expression of high-density transiently transfected
cultures, achieving yields of up to 1 gram per liter of recombinant protein
•
Supports growth and transfection of Expi293F™ cells in culture formats of less
than 1 mL in multi-well plates to greater than 10 L in disposable bioreactors
•
Does not contain phenol red
Chapter 1 Product information
1
Expi293™ Met (-) Expression Medium
Expi293™ Met (-) Expression Medium is an optimized, chemically defined formulation
designed to support methionine labeling of proteins and transfection of 293 cells
(e.g., Expi293F™ cells) in suspension. The medium does not contain methionine,
protein, undefined lysates, or components of animal origin. Expi293™ Met (-)
Expression Medium is a complete, ready-to-use medium formulated with GlutaMAX
Supplement, but requires methionine supplementation. The medium is not
recommended for adherent 293 cell culture.
™
Expi293™ Expression System User Guide
11
Chapter 1
1
Components of the Expi293™ Expression System: structural biology and inducible expression modules
Product information
ExpiFectamine™ 293 Reagent
ExpiFectamine™ 293 Reagent is optimized for the transfection of nucleic acids into
high-density Expi293™ cell cultures.
ExpiFectamine™ 293 Reagent has the following features:
•
Designed specifically for transfection of high-density suspension cell culture,
with matching transfection enhancers that boost transfection performance and
protein expression
•
Achieves protein yields 2- to 10-fold higher than other transfection reagents used
on high-density 293 cell cultures
•
Employs the same transient expression protocols typically used in current lowdensity 293 suspension culture systems to easily switch from low‑density
systems to the high yield, high-density Expi293™ Expression System
•
Provides robust and reproducible transfection results
•
Enables scalable transfections for culture volumes of less than 1 mL to greater
than 10 liters, while maintaining equivalent volumetric protein yields
ExpiFectamine™ 293 Transfection Enhancer 1
ExpiFectamine™ 293 Transfection Enhancer 1 is an optimized, chemically defined,
serum-free, protein-free, animal origin-free formulation designed to work in
conjunction with Expi293™ Expression Medium to support, high-density transient
transfections. ExpiFectamine™ 293 Transfection Enhancer 1 is a component of the
ExpiFectamine™ 293 Transfection Kit.
ExpiFectamine™ 293 Transfection Enhancer 2
ExpiFectamine™ 293 Transfection Enhancer 2 is a proprietary, animal origin-free
formulation developed to be used in conjunction with ExpiFectamine™ 293 Reagent
and ExpiFectamine™ 293 Transfection Enhancer 1 to enhance protein production,
resulting in maximal protein yields. ExpiFectamine™ 293 Transfection Enhancer 2 is a
component of the ExpiFectamine™ 293 Transfection Kit.
ExpiFectamine™ 293 Met (-) Transfection Enhancer 2
ExpiFectamine™ 293 Met (-) Transfection Enhancer 2 is a proprietary, animal originfree, methionine-free formulation developed to be used in conjunction with
ExpiFectamine™ 293 Reagent and ExpiFectamine™ 293 Transfection Enhancer 1 to
enhance protein production during metabolic protein labeling experiments.
ExpiFectamine™ 293 Met (-) Transfection Enhancer 2 is a component of the
ExpiFectamine™ 293 Met (-) Transfection Kit.
12
Expi293™ Expression System User Guide
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