TNT® Eukarayotic Cell-free Protein Expression Systems - Promega

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TNT® Eukarayotic Cell-free Protein Expression Systems - Promega

POI

CELL-FREE PROTEIN EXPRESSION

TnT ® Eukarayotic Cell-free

Protein Expression Systems

The TnT® Systems are convenient

single-tube systems for eukaryotic

cell-free protein expression. To

use these systems 0.2-2.0µg of

circular plasmid DNA containing a

T7, T3 or SP6 promoter, or a PCRgenerated

fragment containing a

T7 promoter is added to an aliquot

of the TnT® Quick Master Mix and

incubated for 60 minutes at 30˚C

(Figure 1). The reaction produces

sufficient quantities of protein that

can be used directly for a variety

of proteomic applications including

protein:protein interactions.

TNT ® Master Mix

Contains

components for

coupled

transcription/

translation

PCR Fragments

or

Plasmid DNA

Add DNA template

containing

sequence of

protein of interest

(POI)

POI

HaloTag

POI

HaloTag

HaloTag

POI

HaloTag

Protein expressed

1-4 HOURS

Use directly for

Application

(Protein:protein

interaction,

Protein:DNA

interaction,

enzyme activity,

others)

Figure 1. Schematic illustrating how the TnT® Quick System functions.

9695MH

Obtain data faster: Functional protein in only one hour, not days as with cell-based expression systems.

Save time: Expressed proteins can be used directly after expression, no requirement for additional purification.

Reliable results: Express functional, soluble protein unlike with E. coli-based systems.

Multiple applications with one system: Use expressed protein for the characterization of protein:protein

interactions, protein:nucleic acid interactions, protein modifications and more.

Ordering Information

PRODUCT SIZE CAT. #

TnT® T7 Quick Coupled Transcription/Translation System

40 reactions L1170

5 reactions L1171

TnT® SP6 Quick Coupled Transcription/Translation System

40 reactions L2080

5 reactions L2081

TnT® SP6 High-Yield Wheat Germ Protien Expression System

40 reactions L3260

10 reactions L3261

TnT® T7 Quick for PCR DNA 40 reactions L5540

TnT is a registered trademark of Promega Corporation.

PROMEGA CORPORATION • 2800 WOODS HOLLOW ROAD • MADISON, WI 53711-5399 USA • TELEPHONE 608-274-4330

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CELL-FREE PROTEIN EXPRESSION

Characterization of Viral

Mediated Diseases

TnT® in vitro Transcription/Translation Systems

References #1

The characterization of viral mediated diseases is critical to promote the overall welfare of humans or

animals. Initial research focused on the interpretation of the genomic content (i.e., DNA- or RNA-based) of

the selected virus. The next step now is to better understand the proteins that are encoded by this content

and their interaction with the host proteome.

The following citations illustrate the use of cell-free protein expression to facilitate this research:

1. Hale, B.G. et al. (2010) J. Virol. 84, 6909-22.

In 2009 an antigenically distinct swine-origin H1N1 influenza

A virus was detected in humans. In order to better

understand the factors that may contribute to replication

efficiency or pathogenicity, the NS1 protein of this virus

was characterized. The H1N1 NS1 protein lacks the ability

to inhibit host gene expression. By generating different

mutated versions of the NS1 protein through the use of

cell-free expression that ability was restored.

2. Dobrikova, E. et al. (2010) J. Virol. 84, 270-9.

Human pathogen virus such herpes simplex viruses manipulate

host cell translation machinery to ensure efficient

expression of viral genes and host cell protein synthesis.

One of the critical factors necessary for the initiation of

host translation is cytoplasmic polyandeylate-binding

protein (PABPC1). PABPC1 simulates eIF4F assembly at

the mGcap and subsequent ribosomal subunit recruitment.

Utilizing cell-free expression to generate proteins for

pull-down assays, several viral proteins that interact with

PABPC1 were identified, resulting in diminished association

of the protein (PABPC1) with the cap binding complex.

3. He, Z. et al. (2010) J. Virol. 84, 2047-62.

Previous studies have demonstrated that the replication

and transcription activator (RTA) encoded by Kaposi’s sarcoma-associated

herpes virus (KSHV) open reading frame

50 plays a pivotal role in control of the virus life cycle.

Using yeast two hybrid screening of a human spleen

cDNA library a novel RTA binding protein was identified,

transducin-like enhancer of split 2 (TLE2). Using cell-free

expression to generate proteins for pull-down assays

three distinct domains of TLE2 that were identified to be

important for interaction with RTA. A similar approach

was used to define the regions of RTA that were required

for interaction with TLE2.

4. Bintintan, I. and Meyers, G. (2010) J. Biol. Chem. 12, 8572-84.

Pestiviruses are positive strand RNA viruses containing a

single open reading frame coding for polyprotein of about

4000 amino acids. The polyprotein is processed by cellular

and viral proteases into 12 mature proteins. Using canine

microsomal membranes in conjunction with cell-free

expression indentified that translocation of protein into

the membranous compartment and subsequent glycosylation

is essential for cleavage.

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CELL-FREE PROTEIN EXPRESSION

Epigenetics-Related

Applications

TnT® in vitro Transcription/Translation Systems

References #2

Epigenetics is the study of processes involved in genetic development of an organism, especially activation/

deactivation of genes. One way genes are regulated is through remodeling of chromatin, the complex of

DNA histone proteins, with which it associates. Conformation of chromatin is profoundly influenced by posttranslational

modification of histone proteins. The following references illustrate the use of cell-free expression

to characterize this process.

1. Shao, Y. et al. (2010) Nucl. Acid. Res. 38, 2813–24.

Carbonic anhydrase IX (CAIX) plays an important role in

the growth and survival of tumor cells. The MORC proteins

contain a CW-type zinc finger domain and are predicted to

have the function of regulating transcription. CAIX mRNA

was shown to be down-regulated 8-fold when MORC2 was

overexpressed. Moreover, MORC2 decreased the acetylation

level of histone H3 at the CAIX promoter. Assays

showed that MORC2 and HDAC4 were assembled on the

same region of the CAIX promoter. Interaction between

MORC2 and HDAC4 were confirmed by using cell free

expression of MORC2 and GST-HDAC (GST pull-downs).

Cell-free expression was also used to express MORC2 proteins

to determine through gel shifts the binding location

on the CAIX promoter region.

2. Denis, H. et al. (2009) Mol. Cell. Biol. 29, 4982–93.

The recent identification of enzymes that antagonize or

remove histone methylation offers new opportunities to

appreciate histone methylation plasticity in the regulation

of epigenetic pathways. PAD4 was the first enzyme

shown to antagonize histone methylation. Through the use

of cell-free expression to express both PAD4 and HDAC1

proteins and E. coli expression of GST fusions of PAD4 and

HDAC1, pulldown experiments confirmed by in vivo experiments

that PADI4 associates with the histone deacetylase

1 (HDAC1).

3. Zhou, R. et al. (2009) Nucl. Acids. Res. 37, 5183–96.

Lymphoid specific helicase (Lsh) belongs to the family

of SNF2/helicases. Disruption of Lsh leads to developmental

growth retardation and premature aging in mice.

However, the specific effect of Lsh on human cellular

senescence remains unknown. In vivo results noted

that Lsh requires histone deacetylase (HDAC) activity to

repress p16INK4a. Moreover, overexpression of Lsh is

correlated with deacetylation of histone H3 at the p16

promoter. In vitro pull-downs using cell free expression

and GST fusions from E. coli were used to collaborate

interactions between Lsh, histone deacetylase 1 (HDAC1)

and HDAC2 observed in vivo.

4. Brackertz, M. et al. (2006) Nucl. Acid. Res. 34, 397-406.

The Mi-2/NuRD complex is a multi-subunit protein complex

with enzymatic activities involving chromatin remodeling

and histone deacetylation. The function of p66α and of

p66β within the multiple subunits has not been addressed.

GST-fused histone tails of H2A, H2B, H3 and H4 were

expressed in E. coli used in an in vitro pull-down assay with

radioactively labeled p66-constructs expressed using cell

free systems. Deletions at the C terminus noted reduced

binding of p66 where as deletions at the N terminus did

not affect binding. Also observed was that acetylation of

histone tails reduces the association with both p66-proteins

in vitro.

PROMEGA CORPORATION • 2800 WOODS HOLLOW ROAD • MADISON, WI 53711-5399 USA • TELEPHONE 608-274-4330

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CELL-FREE PROTEIN EXPRESSION

Plant-Related Applications

TnT® in vitro Transcription/Translation Systems

References #3

Cell-free protein expression can be utilized for the analysis of: protein:protein interactions, protein:nucleic acid

interactions, analysis of post-translational modifications and many other applications. The majority of these

references are based on the characterization of mammalian proteins. However there are several references

using TnT®-based systems (either rabbit reticulocyte lysate or wheat germ-based) for the analysis of proteins

from plants. Examples include:

1. Zhang, Z. et al. (2011) The Plant Cell 23, 273-88.

Using the Beet serve curly top virus C2 protein as bait

in a yeast–two hybrid screen, a C2-interacting protein

S-adenosyl-methionine decarboxylase1 (SAMDC1) was

identified from an Arabidopsis thaliana cDNA library. In

conjunction with other techniques the interaction was

confirmed and characterized using TnT in conjunction

with GST pull-down assays.

2. Gao, M-J. et al. (2009) The Plant Cell 21, 54–71.

The seed maturation program is repressed during germination

and seedling development so that embryonic

genes are not expressed in vegetative organs. Noted in

this reference was evidence that ASIL1 functions as a negative

regulator of a large subset of Arabidopsis embryonic

and seed maturation genes in seedlings. Electrophoretic

mobility shift assays using protein expressed in the TnT

system and labeled DNA oligos corresponding the various

regions of the 2S3 promoter were one of the techniques

used to characterize the specific binding to this

promoter region.

3. Huizinga, D. et al. (2010) Molecular Plant 3, 143–55.

In the characterization of the Arabidopsis farnesycyteine

lysase a protein larger than predicted molecular mass was

observed via gel analysis. In order to determine if this was

due to N-glycosylation of the protein, the FC lyase protein

was synthesized using the TnT system in the presence or

absence of canine pancreatic microsomal membranes.

4. Tanz, S. et al. (2009) Plant Physiology 150, 1515-29.

Carbonic anhydrase catalyzes the reversible hydration of

CO2 and is involved in both C(3) and C(4) photosynthesis,

however its role and intercellular and intracellular locations

differ between C(3) and C(4) plants. Three different

cDNA encoding distinct β-carbonic anhydrases were

isolated from leaves of the C(3) plant Flaveria pringlei. To

determine if either of the proteins encoded by the cDNA

clones are targeted for chloroplasts. Labeled proteins

were expressed using the TnT system and subjected to

chloroplast protein import analysis.

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CELL-FREE PROTEIN EXPRESSION

Protein Arrays

TnT® in vitro Transcription/Translation Systems

References #4

The traditional methods of generating protein arrays require separate expression of hundreds of proteins,

followed by purification and immobilization of the proteins on a solid surface. Cell-free protein array

technology produces protein microarrays by performing in vitro synthesis of the target protein from their DNA

templates.

One approach for the generation of cell-free-based microarrays is the nucleic acid programmable protein array

(NAPPA). NAPPA uses DNA template that is biotinylated and is bound to avidin that is pre-coated onto the

protein capture surface. Newly synthesized proteins which are tagged with GST are then immobilized next to

the template DNA by binding to an adjacent polyclonal anti-GST capture antibody (Figure 1).

The following references, illustrate the use of

NAPPA to screen hundreds of proteins.

GST-tagged protein

1. Wright, C. et al. (2011) Mol. Cell. Biol. (in press).

To determine the extent of autoantibodies

expression in the case of ankylosing spondylitis,

AS (a common inflammatory disease) plasmid

DNA representing 3498 proteins was arrayed and

expressed then screened against with sera from

patients with anklosing spondylitis and healthy

controls. Results indicated that AS patients’ auto

response were targeted towards connective,

skeletal and muscular tissue.

2. Anderson, K. et al. (2010) J. Proteomics Res. 10,

85-96. 4988 ORF candidate tumor antigens were

arrayed as plasmid DNA and proteins expressed and

captured on a protein microarray using the NAPPA

technology. Replicate arrays were probed with

sera from patients with breast cancer and healthy

controls. In conjunction with other assays a panel of

28 biomarkers were determined.

TNT ® GST

Coupled

Transcription/

Translation

System

GST GST

B B B

Anti-GST

antibody

Avidin

GST

GST

Biotinylated

plasmid DNA

Figure 1. Nucleic Acid Programable Protein Array (NAPPA).

9982MB


CELL-FREE PROTEIN EXPRESSION

TnT ® in vitro Transcription/Translation Systems

References #4

Cell-Free Protein Expression: Protein Arrays

3. Rolf, A. et al. (2008) Proc. Natl. Acad. Sci. 105,

4364-69. In this reference they report the assembly

of a complete ORF collection (3887) for V. cholerae.

15% of this collection was transferred into relevant

GST expression vectors and arrayed as plasmid DNA

and subsequently expressed using Promega’s TnT

system. The resulting proteins were screened to

determine if they triggered a Toll-like recepetors

(TLR) using A549 cells containing a luciferase gene

transcribed under control of a NF-κβ response

promoter. Using this approach a novel TLR5 agonist

(FlaC) was characterized.

4. Motor, W. et al. (2009) Infection Immun. 77, 4877-

86. A comprehensive study of 266 gene products

of the 5570 ORFs of Psuedmonas aeruginosa that

were likely to reside in the outer membrane or

secreted into the surrounding media were screened

(using the NAPPA technology) for their antigenicity.

From this study it was possible to identify 12

proteins that trigger an adaptive immune response

in cystic fibrosis and acutely infected patients.

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CELL-FREE PROTEIN EXPRESSION

One System,

Multiple Applications

TnT® in vitro Transcription/Translation Systems

References

Promega TNT® Coupled Transcription/Translation Systems offer a one step, easy to perform protocol with

significant time savings over cellular approaches to protein expression. Proteins expressed using these systems

can be used for a variety of applications relating to their relationship with other molecules and function.

References

Protein Pull-downs

Ma, W. et al. (2010) J. Virol. 84,

2122-33.

Kim, S.M. et al. (2010) Nucl. Acid.

Res. 38, 6389-403.

Camacho, L. et al. (2009) J. Cell.

Sci. 122, 4383-92.

Houshmandi, S. et al. (2009) Mol.

Cell Biol. 29, 1472-86.

Gel Shift (EMSA)

Buas, M. et al. (2010) J. Biol.

Chem. 285, 1249-58.

Lin, Y. et al. (2009) Drug. Metab.

Dispos. 37, 1295-304.

Bollig, F. et al. (2009) Develop.

136, 2883-92.

Chloroplast Import Assays

Tanz, S. et al. (2009) Plant. Phy.

150, 1515-29.

Co-immunoprecipitation

Xi, Gang. et al. (2010) J. Biol.

Chem, 285, 6937-51.

Martin, L. and Tremblay, J.

(2009) J. Mol. Endocrinol. 42,

119-29.

Makarenkova, H. et al. (2009) J.

Biol. Chem. 284, 14866-74.

Marchetti, B. et al. (2009) J. Gen.

Virol. 90, 2865-70.

Ubiquitination Assays

Cen, B. et al. (2010) J. Biol. Chem.

285, 29128-37.

Zhong, B. et al. (2010) J. Immunol.

184, 6249-55.

Fu, W. et al. (2009) J. Biol. Chem.

284, 13987-14000.

Protein Folding

Berry, J-L and Bulleid, N. (2009)

BioSci. Horz. 2, 13-21.

Sumoylation Assays

Rytinki, M. et al. (2009) J. Biol.

Chem. 284, 26184-93.

Klein, U. et al. (2009) Mol. Cell.

Biol. 20, 410-18.

Seo, W. and Ziltener, H. (2009)

Blood, 17, 3567-77.

Inhibition of Translation

Zhang, Y. and Inouye, M. (2009) J.

Biol. Chem. 284, 6627-38.

Liu, Y. et al. (2009) J. Biol. Chem.

284, 5859-68.

Membrane Association

Bintintan, I. and Meyers, G. (2010)

J. Biol. Chem. 285, 8572-84.

Al, L-S. et al. (2009) J. Virol. 83,

9923-39.

Stapleford, K. et al. (2009) J. Virol.

83, 4498-507.

PROMEGA CORPORATION • 2800 WOODS HOLLOW ROAD • MADISON, WI 53711-5399 USA • TELEPHONE 608-274-4330

www.promega.com • ©2010 PROMEGA CORPORATION • ALL RIGHTS RESERVED • PRICES AND SPECIFICATIONS SUBJECT TO CHANGE WITHOUT PRIOR NOTICE • PRINTED IN USA, REV 3/11 • 19506 • PART #FL254

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