Figure 1. Typical N-linked Glycan Processing for Immunoglobulins

Man5 is the predominate glycan pattern in Expi293 GnTI (N-acetylglucosaminyltransferase I)

deficient cells.

AbstractMembrane proteins are a notoriously difficult class of proteins to express, purify

and characterize. Expression levels of membrane proteins in mammalian

expression systems, the preferred system to generate native-like post-

translational modifications, are often so low that yields are unsuitable for the

intended downstream uses. Here, we present case studies on the optimization of

ion channel and GPCR expression using the Expi293 and ExpiCHO Expression

Systems. Additionally, we demonstrate the utility of a new range of Expi293

reagents, including engineered Expi293 cell lines (e.g. GNTI-, Inducible and

Inducible/GNTI- Expi293 cell lines) to allow for regulated expression and/or

glycosylation of membrane proteins. Together, these protein expression tools

significantly enhance the ability of researchers to study membrane protein

biology.

For Research Use Only. Not for use in diagnostic procedures. Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com

Optimized Expression of Membrane Proteins in the Expi293 and ExpiCHO

Expression Systems: New Tools for Difficult to Express Proteins

Chao Yan Liu1, Jian Liu1, Wanhua Yan1, Sam Stepnowski1 ,Kyle Williston1, Katy Irvin1, Chetana Revankar2, Natasha Lucki2, Henry Chiou2,

Jonathan Zmuda1. Thermo Fisher Scientific, 1Frederick, MD, U.S.A, 2Carlsbad, CA, U.S.A;

Corresponding Author: Jon.Zmuda@Thermofisher.com

I. Expi293 GnTI(-) Cell Line

Expi293 reagents to support hard-to-express

proteins and structural biology applications• Expi293 GnTI(-) Cell Line

• Expi293 Inducible Cell Line

• Expi293 Inducible GnTI(-) Cell Line

• Expi293 Methionine-Deficient Labeling

By Dna 621 - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=31825198

Man5

Man5

G2F

G1F

G0F

M3N2F+GlcNac

Figure 3. Glycan Patterns for Two Different Proteins Expressed in the Expi293

GnTI(-) Cell Line.

(A) Glycan patterns for human IgG expressed in Expi293 and (B) Expi293 GnTI(-) cell

lines. Greater than 98% of glycans expressed by Expi293 GnTI(-) cells are of the

Man5 variety. (C) Expression of the GPCR CB2 in Expi293 (Lanes 1-4) and Expi293

GnTI(-) (Lanes 5-8) cells under varying expression conditions.

Figure 2. Growth and Expression Profiles of Expi293 GnTI(-) cells

(A) Growth kinetics of Expi293 GNTI(-) cells are comparable to parental Expi293 cells. (B)

Expi293 GnTI(-) cells express proteins at levels comparable to parental Expi293 cells. By

comparison, expression levels of ATCC GnTI(-) cells adapted into FreeStyle293 media with PEI

transfection are >30-fold lower than Expi293 GnTI(-) cells in the Expi293 System.

II. Expi293 Inducible Cell Line

Days in Culture

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Expi293 Inducible Expi293 Control

Expi293 Inducible Expi293 Control

Figure 6. Modulation of Protein Expression in Expi293 Inducible Cells

(A) Cells were transfected with an Fc-fusion protein in pcDN5.0/TO vector and then

induced with different levels of tetracycline in a dose-dependent manner. (B, C) CB2-GFP

GPCR expression with different levels of induction.

III. Expi293 Inducible GnTI(-) Cell Line

Days in Culture

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Inducible GnTi- Expi293 Control

Inducible GnTi-T Expi293 Control

Figure 7. Growth and Expression Profiles of Expi293 Inducible GnTI(-) cells

(A) Growth kinetics of Expi293 Inducible GnTI(-) cells are comparable to parental Expi293 cells.

(B) Upon full induction in pcDNA5.0/TO vector, Expi293 Inducible GNTI(-) cells express

proteins at levels comparable to parental Expi293 cells. By comparison, expression levels of T-

Rex™ 293 cells adapted into FreeStyle293 media with PEI transfection are 8-fold lower than

the Expi293 Inducible GnTI(-) cell line in the Expi293 System.

Figure 8. Glycan Patterns for Human IgG and Erythropoietin Expressed in the

Expi293 Inducible GnTI(-) Cell Line.

(A) Glycan patterns for human IgG and erythropoietin (epo) expressed in Expi293 and

(B) Expi293 Inducible GnTI(-) cell lines. (C) SDS PAGE of: 1) native epo, 2) epo

expressed in the Expi293 Inducible GnTI(-) cell line, 3) PNGaseF deglycosylated epo

from Expi293, 4) deglycosylated epo from Expi293 Inducible GnTI(-) cells.

A B

A B

Days in Culture

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Expi293 GnTi- Expi293 Control

Expi293 GnTi- Expi293 Control

IV. Optimization of AA2AR GPCR Expression

Figure 10. Optimization of GPCR expression in the Expi293, Expi293 GnTI(-),

and ExpiCHO expression systems

(A) AA2AR GPCR expression was optimized by titrating plasmid DNA, Feed and Enhancers in

Expi293, Expi293 GnTI(-), and ExpiCHO cells. AA2AR was harvested on days 2 and 3 post-

transfection and analyzed by FACS. The mean fluorescence intensity (MFI) was multiplied by

the viable cell density (VCD) at the time of harvest. The data revealed significant factors for

each expression system.

BA

A

B

C Expi293 Expi293 GNTI(-)

1 2 3 4 5 6 7 8

Figure 5. Growth and Expression Profiles of Expi293 Inducible cells

(A) Growth kinetics of Expi293 Inducible cells are comparable to parental Expi293 cells. (B)

Upon full induction in pcDNA5.0/TO vector, Expi293 Inducible cells express proteins at levels

comparable to parental Expi293 cells. By comparison, expression levels of T-Rex™ 293 cells

adapted into FreeStyle293 media with PEI transfection are nearly 10-fold lower than the

Expi293 Inducible cell line in the Expi293 System.

Figure 4. Principles of Inducible Protein Expression

Expi293 cells were stably transfected to express high levels of the TET repressor protein.

In the absence of induction, TET repressor protein binds to the TET operator sequence of

the plasmid and prevents expression of the protein of interest.

Figure 9. Modulation of Protein Expression in Expi293 Inducible GnTI(-) Cells

(A). Cells were transfected with a plasmid encoding an Fc-fusion protein in pcDN5.0/TO vector

and then induced with different levels of tetracycline. (B,C) CB2-GFP GPCR expression with

different levels of induction.

>30X>8X

A

B

C

Man5G2F

G1F

G0F

M3N2F+GlcNac

A B

98

62

49

38

28

14

Endoplasmic

ReticulumGolgi

>9X

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1 2 43

0.010 0.03 0.05 0.1

BF

GFP

Tetracycline concentration (mg/mL)

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2 1 05

T e t r a c y c l i n e c o n c e n t r a t i o n ( m g / m L )

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T e t r a c y c l i n e c o n c e n t r a t i o n ( m g / m L )

Tetracycline concentration (mg/mL)

0 0.03 0.05 0.1 1

BF

GFP

C

C

A

Figure 12. AA2AR expression in Expi293 Inducible cells

VI. Expression of AA2AR in Expi293 Inducible Cells

(A) AA2AR expression in Expi293 cells or Expi293 Inducible cells. Expi293 inducible

cells were induced at two time points along with feed and enhancer. AA2AR was

harvested on day 2 and 3 post-transfection for Expi293 cells and day 2 and 3 post-

induction for Expi293 Inducible cells. The mean fluorescence intensity (MFI) was

multiplied by the viable cell density (VCD) at the time of harvest. Inset: MFI, not

considering viable cell density.

Figure 11. AA2AR GPCR expression levels in the Expi293, Expi293 GnTI(-), and

ExpiCHO expression systems

(A) AA2AR GPCR optimized expression in the Expi293 Expression System; significant

factors for optimized expression were DNA (0.5mg/mL) and enhancer. (B) AA2AR GPCR

optimized expression in the Expi293 GnTI(-) Expression System; significant factors for

optimized expression were DNA (1.0mg/mL) and enhancer. (C) AA2AR GPCR optimized

expression in the ExpiCHO Expression System; significant factors for optimized expression

were feed and enhancer.

A B

C

* Catalog Protocol at 37C

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