Cell-Free Protein Expression Systems

21 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

3.1 Translation Systems: mRNA-based 25

Rabbit Reticulocyte Lysate System, Nuclease-Treated 26

Flexi

Rabbit Reticulocyte Lysate System 27

Wheat Germ Extract 28

3.2 Transcription and Translation Systems: DNA-based 29

Rabbit Reticulocyte Lysate Systems

TnT

SP6 Coupled Reticulocyte Lysate System 31

TnT

T7 Coupled Reticulocyte Lysate System 31

TnT

T3 Coupled Reticulocyte Lysate System 31

TnT

T7 Quick Coupled Transcription/Translation System 31

TnT

SP6 Quick Coupled Transcription/Translation System 31

TnT

T7 Quick for PCR DNA 31

Wheat Germ Extracts

TnT

SP6 High-Yield Wheat Germ Protein Expression System 33

Insect Cell Lysate System

TnT

T7 Insect Cell Extract Protein Expression System 34

E. coli Extracts

E. coli S30 Extract System for Linear Templates 35

S30 T7 High-Yield Protein Expression System 36

3.3 Cell-Free Protein Labeling Reagents 37

FluoroTect

™

Green

Lys

in vitro Translation Labeling System 38

Transcend

™

Non-Radioactive Translation Detection Systems 39

3.4 Membrane Vesicles for Signal Peptide Cleavage

and Core Glycosylation 40

Canine Pancreatic Microsomal Membranes 41

22 Discover Reliable Tools for Protein Analysis

Introduction

Cell-free protein synthesis is an important tool for

molecular biologists in basic and applied sciences. It

is increasingly being used in high-throughput functional

genomics and proteomics, with significant advantages

compared to protein expression in live cells. Cell-free

protein synthesis is essential for the generation of

protein arrays, such as nucleic acid programmable

protein array (NAPPA) and enzyme engineering using

display technologies. The cell-free approach provides

the fastest way to correlate phenotype (function of

expressed protein) to genotype. Protein synthesis can be

performed in a few hours using either mRNA template in

translational systems or DNA template (plasmid DNA or

PCR fragments) in coupled transcription and translation

systems. Furthermore, cell-free protein expression

systems are indispensable for the expression of toxic

proteins, membrane proteins, viral proteins and for

proteins that undergo rapid proteolytic degradation by

intracellular proteases.

23 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

Origins of Cell-Free Expression Systems

Cell-free protein expression lysates are generated from cells engaged in a high rate of protein

synthesis, such as immature red blood cells (reticulocytes). The most frequently used cell-free

expression systems originate from rabbit reticulocytes, wheat germ and E. coli. There are two

types of cell-free expression systems: Translation Systems and Coupled Translation and

Transcription (

TNT

) Systems (Figure 3.1). Both types of systems provide the macromolecular

components required for translation, such as ribosomes, tRNAs, aminoacyl-tRNA synthetases,

initiation, elongation and termination factors. To ensure efficient translation, each extract has to be

supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems and

salts (Mg

, K

, etc.). For eukaryotic systems creatine phosphate and creatine phosphokinase

serve as energy regenerating system, whereas prokaryotic systems are supplemented with

phosphoenol pyruvate and pyruvate kinase. Coupled transcription and translation systems are

supplemented with phage-derived RNA polymerase (T7, T3 or SP6) allowing the expression of

genes cloned downstream of a T7, T3 or SP6 promoter.

Selection of Cell-Free Protein Expression

Many different cell-free expression

systems derived from prokaryotic and

eukaryotic source are available. The

choice of the system is dependent on

several factors, including the origin of

the template RNA and DNA, protein

yield or whether the protein of interest

requires post-translational modification

(e.g., core glycosylation). We offer

translation systems (mRNA-based)

and coupled transcription/translation

systems (DNA-based) from prokary-

otic and eukaryotic sources. Table 3.2

provides an overview of translational

systems and Table 3.3 provides an

overview of coupled translation/tran-

scription systems.

Figure 3.1. Cell-free protein expression systems are divided into mRNA-based

translation systems and in DNA-based transcription/translation systems.

Plasmid

DNA

PCR Fragments

Cell-Free Translation

Systems

Cell-Free Transcription/

Translation Systems

mRNA

Protein

RNA

12176MA

24 Discover Reliable Tools for Protein Analysis

Table 3.1. Applications of Cell-Free Protein Synthesis

Functional Genome/

Proteome Analysis

•

Gene mutation/deletion analysis

(e.g., enzyme kinetics)

•

Protein domain mapping

•

Characterization of protein interactions

•

Gel Shift EMSA

•

Generation of protein arrays

Expression of Difficult-to-

Express Proteins

•

Cell-toxic proteins, membrane protein,

viral proteins, kinases

Protein Evolution/

Enzyme Engineering

•

Display technologies (e.g., ribosome,

mRNA display, in vitro compartmental-

ization)

•

Evolution of antibodies in vitro by

ribosome display

Analysis of Transcriptional/

Translational Regulation

•

Structural RNA analysis such as char-

acterization of regulatory elements for

translation (e.g., UTRs, Capping, IRES)

•

RNA virus characterization

Screenings

•

Screening of chemical libraries for

effect on translation

•

Drug screening (e.g., antibiotics)

Protein Labeling

•

Open systems allow

the incorporation of

labeled amino acids

25 Discover Reliable Tools for Protein Analysis

Table 3.2. Overview of Cell-FreeTranslation Systems that use mRNA as a Template.

Translation

System

Nuclease-

Treated

Signal Cleavage &

Core Glycosylation

with CMM*

Labeling

Options**

Luciferase

Control RNA

Protein

Yield

Rabbit Reticulocyte

Lysate System,

Nuclease-Treated

(Cat.# L4960)

+ +

Met,Cys,Leu,

FluoroTect

™

;

Transcend

™

+ 1–4 µg/ml

Flexi

Rabbit

Reticulocyte Lysate

(Cat.# L4540) ***

+ +

Met, Cys, Leu,

FluoroTect

™

;

Transcend

™

+ 1–4 µg/ml

Wheat Germ Extract

(Cat.# L4380)

+ -

Met, Cys, Leu,

FluoroTect

™

;

Transcend

™

+ 0.6–3 µg/ml

* CMM: Canine Microsomal Membranes

** The lysates are provided with three Amino Acid Mixtures for the incorporation of labeled amino acids like methionine, cysteine & leucine. Transcend

™

tRNA

(Cat.# L5070; L5080) and FluoroTect

™

(Cat.# L5001) can be used to incorporate biotinylated or fluorescently labeled lysine residues.

*** The system provides greater flexibility of reaction conditions than standard rabbit reticulocyte lysate systems. The Flexi

Rabbit Reticulocyte Lysate System

allows translation reactions to be optimized for a wide range of parameters, including Mg

and K

concentrations and the option to add DTT.

Cell-free translation systems are used for protein

expression of either in vitro transcribed mRNA or mRNA

isolated from tissues or cells. These systems are used

to express single proteins as well as multiple proteins

in high-throughput applications such as display tech-

nologies. Furthermore, cell-free translation systems are

useful for functional and structural RNA analysis, or to

study aspects of the translational machinery. Eukaryotic

translation systems originate from either rabbit reticu-

locyte lysates (RRL) or wheat germ extracts (WGE).

We offer three mRNA-based translation systems. The

extracts are treated with microccal nuclease to destroy

endogenous mRNA and thus reduce background translation

to a minimum (Table 3.2).

The Flexi

Rabbit Reticulocyte Lysate System offers greater

flexibility in reaction conditions by allowing translation

reactions to be optimized for a wide range of parameters,

including Mg

and K

concentrations. The Wheat Germ

Extract is a useful alternative to the RRL systems for

expressing small proteins or for expressing proteins known

to be abundant in RRL. Researchers expressing proteins

from plants or yeasts or other fungi also may find WGE

preferable to RRL.

3.1 Translation Systems: mRNA-based

OVERVIEW

Cell-Free Protein

Expression Systems

26 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

Rabbit Reticulocyte Lysate System,

Nuclease-Treated

In vitro protein synthesis starting from mRNA.

Description

Rabbit Reticulocyte Lysate (RRL), Nuclease-Treated, is optimized for mRNA translation by the addition of several supple-

ments. These include hemin, which prevents activation of the heme-regulated eIF-2a kinase; an energy-generating system

consisting of phosphocreatine kinase and phosphocreatine; and calf liver tRNAs to balance the accepting tRNA popula-

tions, thus optimizing codon usage and expanding the range of mRNAs that can be translated efficiently. In addition, the

lysates are treated with micrococcal nuclease to eliminate endogenous mRNA. RRLs post-translationally modify proteins via

phosphorylation, acetylation and isoprenylation. Signal peptide cleavage and core glycosylation also can be achieved by the

addition of Canine Pancreatic Microsomal Membranes. See Table 3.1 for additional applications.

Ordering Information

Rabbit Reticulocyte Lysate (RRL),

Nuclease-Treated (Cat.# L4960)

Principle

In RRL translation reactions, mRNA is used as template

for translation. In general, optimal results will be achieved

after an incubation time of 1.5 hours at 30°C. However,

many template-related factors affect translation efficiency

of specific mRNAs in the RRL system and should be

considered when designing in vitro translation experiments.

The optimal mRNA concentration will vary for particular

transcripts and should be determined empirically. In

addition, the presence of certain nucleic acid sequence

elements can have profound effects on initiation fidelity and

translation efficiency. Poly(A)+ tails, 5´-caps, 5´-untranslated

regions and the sequence context around the AUG start (or

secondary AUGs in the sequence) all may affect translation

of a given mRNA.

Features and Benefits

• Consistent: Reliable and consistent translation with each

lot.

• Optimized and Ready-to-Use: The treated Rabbit

Reticulocyte Lysate is optimized for translation.

• Convenient: Luciferase Control RNA included.

Figure 3.2. Flow chart of in vitro translation procedure using Rabbit

Reticulocyte Lysate.

Translation Systems: mRNA-based

mRNA

Protein

mRNA

12146MB

Rabbit

Reticulocyte

Lysate

Incubate for

1.5 hour at 30°C.

Synthesize RNA in vitro or

Isolate mRNA from tissue cells

Analyze with activity assays or protein detection.

27 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

Translation Systems: mRNA-based

Flexi

Rabbit Reticulocyte Lysate System

In vitro protein synthesis starting from mRNA. Optimize translation for

low-expressing mRNA.

Description

The Flexi

Rabbit Reticulocyte Lysate System is widely used to identify mRNA species and characterize their products.

It provides greater flexibility of reaction conditions than the Rabbit Reticulocyte Lysate, Nuclease-Treated, by allowing

translation reactions to be optimized for a wide range of parameters, including Mg

and K

concentrations. See Table 3.1

for additional applications.

Ordering Information

Flexi

Rabbit Reticulocyte Lysate

System (Cat.# L4540)

Principle

As with the standard Rabbit Reticulocyte Lysate, the

Flexi

Rabbit Reticulocyte Lysate System is optimized

for translation by addition of the following supplements:

hemin, to prevent inhibition of initiation factor eIF-2α;

an energy-generating system consisting of pretested

phosphocreatine kinase and phosphocreatine; calf liver

tRNAs to balance the accepting tRNA populations, thus

optimizing codon usage and expanding the range of

mRNAs that can be translated efficiently; and micro-

coccal nuclease to eliminate endogenous mRNA, thus

reducing background translation. This eukaryotic system

has been reported to post-translationally modify proteins

via phosphorylation, acetylation and isoprenylation.

With the addition of Canine Pancreatic Microsomal

Membranes signal peptide cleavage and core glycosyl-

ation can occur. The Flexi

Rabbit Reticulocyte Lysate

System provides greater flexibility of reaction conditions

than standard RRL systems.

Features and Benefits

• Consistent: Reliable and consistent translation with

each lot.

• Easy Optimization: To aid in optimizing magnesium

concentrations, the endogenous magnesium

concentration is provided for each lot of Flexi

Lysate.

• Convenient: Luciferase Control RNA and detection

reagent included.

28 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

Translation Systems: mRNA-based

Wheat Germ Extract

In vitro protein synthesis starting from mRNA.

Description

Wheat Germ Extract (WGE) is a well-defined processed and optimized extract from wheat germ. It contains the cellular

components necessary for protein synthesis (tRNA, ribosomes and initiation, elongation and termination factors). The extract

is supplemented with an energy-generating system (phosphocreatine/phosphocreatine kinase), and with spermidine to

stimulate the efficiency of chain elongation. Only exogenous amino acids and mRNA are needed to initiate translation.

Potassium acetate can be used to optimize translation for a wide range of mRNAs. See Table 3.1 for additional applications.

Principle

Wheat Germ Extract is a useful alternative to the Rabbit

Reticulocyte Lysate (RRL) systems for expressing small

proteins or for expressing proteins expected to be

abundant in RRL. Researchers expressing proteins from

plants, yeast or other fungi also may find Wheat Germ

Extract preferable to RRL.

Features and Benefits

• Optimized Extract: Assists in expression of eukaryotic

messages that do not express well in RRL.

• Flexible: Three Amino Acid Mixtures are provided, which

enable different radioisotope choices.

• Robust: Potassium Acetate is provided to enhance

translation for a wide range of mRNAs.

• Convenient: Luciferase Control RNA included.

Ordering Information

Wheat Germ Extract (Cat.# L4380)

29 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

29 Discover Reliable Tools for Protein Analysis

Coupled transcription and translation (TNT

) systems

offer researchers time-saving alternatives for eukary-

otic in vitro transcription and translation, by coupling

these processes in a single tube format.

TNT

Systems

are used for a variety of applications in low- to high-

throughput functional genome and proteome analyses,

as summarized in Table 3.1.

TNT

Systems are supple-

mented with T7, T3 or SP6 RNA polymerases, allowing

protein expression from DNA cloned downstream of a T7,

T3 or SP6 promoter.

We offer

TNT

Systems originating from eukaryotic

sources such as rabbit reticulocyte, wheat germ and

insect cells as well as from prokaryotic E. coli extracts

(Table 3.3).

The highest production rates are normally achieved

with E. coli extracts. However, eukaryotic systems

often produce eukaryotic proteins with higher activity.

Therefore, the origin of the protein of interest should be

considered when selecting a cell-free expression system.

DNA Template Consideration: Plasmids and

PCR-Fragments

The performance of cell-free systems depends on the

DNA template used. Basically, any vector containing T7,

SP6 or T3 promoters can be used with

TNT

Systems.

However, there are several points to consider when engi-

neering a DNA fragment or plasmid for optimal expres-

sion in a eukaryotic system: (i) the ATG start codon

in the sequence should be the first ATG encountered

following the transcription start site; (ii) ideally, following

the promoter, the ATG is included in a Kozak consensus

sequence; (iii) a stop codon should be included at

the 3´- terminus of the sequence; and (iv) a synthetic

poly(A) tail should be included following the stop codon.

Additionally, vectors used in the

TNT

T7 Coupled Wheat

Germ System should either be linearized or have a T7

transcription terminator in a circular template.

In prokaryotic systems, the selection of a start codon gener-

ally depends on the presence of a ribosomal binding site

(RBS; Shine-Dalgarno sequence), which contains a signal

that marks the start of the reading frame. The presence of

an optimal RBS can greatly increase expression in prokary-

otic systems. The prokaryotic system does not recognize

ATGs upstream of the ATG start codon unless they contain

a properly positioned RBS.

Promega vectors approved for use with

TNT

Systems can

be found in Table 9.1.

The template considerations mentioned above are also valid

for using PCR fragments as templates for the

TNT

reaction.

For the generation of the PCR fragments for protein expres-

sion in eukaryotic systems, the integration of a Kozak

sequence downstream of a T7 or SP6 promoter is recom-

mended (Figure 3.4).

Labeling of Proteins during in vitro Synthesis

All TNT

Systems are provided with three different Amino

Acid Mixtures for the incorporation of radiolabeled amino

acids like methionine, cysteine and leucine. Transcend

™

tRNA and FluoroTect

™

Systems can be used to incorporate

biotinylated or fluorescently-labeled lysine residues (see

Section 3.3).

Signal Peptide Cleavage and Core Glycosylation

Rabbit reticulocyte lysate has been reported to post-transla-

tionally modify proteins via phosphorylation, acetylation and

isoprenylation. However, the addition of Canine Pancreatic

Microsomal Membranes (CMM), to RRLs is required to

achieve signal peptide cleavage and core glycosylation of

the translation product.

3.2 Transcription and Translation Systems: DNA-based

OVERVIEW

30 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

30 Discover Reliable Tools for Protein Analysis

Table 3.3. Overview of Transcription and Translation Systems

NR: Not Recommended

DNA templates for TNT

E.coli Systems requires the Shine Dalgarno ribosomal binding site (RBS).

DNA templates for eukaryotic TNT

Systems should preferably contain the Kozak consensus sequence for translation initiation.

CMM: Canine Microsomal Membranes.

Control DNA contains the firefly luciferase gene. Luciferase activity is detected by the Luciferase Assay Reagent (Cat.# E1500).

Translation reactions can be further optimized by adding Mg

and K

SP6 circular plasmids give higher yields than T7 or T3 circular plasmids; T7 or T3 linearized plasmid may be considered as templates; SP6 linearized plasmids

are not recommended.

Not recommended for SP6 containing template.

For T7 circular plasmids include the T7 terminator sequence; otherwise linearized plasmids are preferred; for SP6 templates only circular plasmids.

Only linearized templates.

Rabbit

TnT

Coupled Reticulocyte Lysate

System (T7, T3, or SP6 RNA

Polymerase; Cat.# L4610, L4950,

L4600)

– +

Met, Cys, Leu,

FluoroTect

™

Transcend

™

+ + 3–6µg/ml

TnT

Quick Coupled Transcription/

Translation (T7 or SP6 RNA

Polymerase; Cat.# L1170, L2080)

– +

Met,

FluoroTect

™

Transcend

™

+ + 3–6µg/ml

TnT

T7 Quick for PCR DNA

(Cat.# L5540)

NR + – +

Met,

FluoroTect

™

Transcend

™

+ – 3–6µg/ml

Wheat

Germ

TnT

Coupled Wheat Germ (T7 or

SP6 RNA Polymerase)

(Cat.# L4130, L4140)

– +

Met, Cys, Leu,

FluoroTect

™

Transcend

™

– + 3–6µg/ml

TnT

SP6 High-Yield Wheat Germ

Protein Expression System (Cat.#

L3260)

+ + – +

Met, Cys, Leu,

FluoroTect

™

Transcend

™

– – 10–100µg/ml

Insect

TnT

Insect Cell Extract Protein

Expression System (Cat.# L1101)

+ NR – +

Met, Cys, Leu,

FluoroTect

™

Transcend

™

–

Control

DNA

15–75µg/ml

E. coli

E coli S30 for Linear DNA

(Cat.# L1030) relies on

endogenous RNA polymerases

+ + –

Met, Cys, Leu,

FluoroTect

™

Transcend

™

– + 1–5µg/ml

S30 T7 High-Yield Protein

Expression System (Cat.# L1110)

+ NR + –

Met, Cys, Leu,

FluoroTect

™

Transcend

™

–

Control

DNA

200–500µg/ml

System

Control DNA &

Detection Reagent

Labeling Options

Plasmid DNA (Circular

or Linearized)

Signal cleavage &

glycosylation with CMM

RBS Required

Kozak Preferred

PCR-generated DNA

Yield

31 Discover Reliable Tools for Protein Analysis

TnT

Coupled Reticulocyte Lysate Systems

Robust eukaryotic cell-free expression systems for the expression of functional

mammalian proteins in a simple one-step procedure.

1537MD

TNT

Quick Master Mix

Add T

RNA Polymerase

Add T

RNA Reaction Buffer

Rabbit Reticulocyte Lysate.

1. Add label of choice.

2. Add DNA template and

Nuclease-Free Water.

3. Inubate at 30

∘

C for

60–90 minutes.

4. Separate translation

products by SDS-PAGE.

Add Amino Acid Mixture Minus Methionine.

Add RNasin

Ribonuclease Inhibitor

TNT

Coupled

Reticulocyte

Lysate System

Detect

TNT

Quick

Coupled

Transcription/

Translation

System

Less Time,

Less Handling!

Figure 3.3. Comparison of the TNT

Coupled Reticulocyte Lysate System and the TNT

Quick Coupled Transcription/Translation System protocols.

Description and Principle

We offer two types of Rabbit Reticulocyte Lysate

Transcription and Translation (

TNT

) Systems: The TNT

Coupled (T7, T3, SP6) System and the

TNT

Quick

Coupled (T7, SP6) System. The main difference between

these systems is that the

TNT

Quick Coupled System

provides a master mix containing all the reaction compo-

nents required in one tube, whereas the

TNT

Coupled

System has all the reaction components provided in

separate tubes (Figure 3.3).

TNT

T7 Quick for PCR DNA

is a rapid and convenient coupled

TNT

System designed

for expression of PCR-generated DNA templates. The

system is robust and able to express a variety of proteins

ranging in size from 10–150kDa. The lysates are supplied

with all reagents needed for

TNT

reactions including RNA

Transcription and Translation Systems: DNA-based

Cell-Free Protein

Expression Systems

polymerases. To use these systems, DNA is added directly

TNT

Lysate and incubated in a 50µl reaction for 60–90

minutes at 30°C. See Table 3.1 for additional applications.

Features and Benefits

• Use in Multiple Applications: The TNT

Systems are

widely used for functional genomics and proteomics

analyses.

• Save Time: The

TNT

Reaction is completed in a maxi-

mum of 1.5 hours.

• Complete System: All reagents for the

TNT

Reaction

are provided (except for labeled amino acids).

• Reliable: Can eliminate solubility issues by using an

in vitro mammalian system.

32 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

Transcription and Translation Systems: DNA-based

2802TA

1 2 3 4 5 1 2 3 4 5

TNT

T7 Quick TNT

T7 Quick for PCR

Vendor A T

T7 Quick for PCR

Vendor B T

T7 Quick for PCR

Figure 3.5. TNT

T7 Quick for PCR was used to express variants of

the APC gene and BRCA1 gene. PCR fragments were used as starting

material for the

TNT

reaction. Transcend

™

tRNA was included in the

reaction for the incorporation of biotinylated lysine residues. Lane 1

contains the no DNA controls; lane 2, APC Seg 2 PCR fragment; lane

3, APC Seg 3 PCR DNA fragment; lane 4, BRCA1 Seg 3 PCR fragment;

lane 5, the Luciferase T7 Control DNA.

Ordering Information

TNT

Coupled Reticulocyte Lysate Systems:

TNT

SP6 Coupled Reticulocyte Lysate System

(Cat.# L4600)

TNT

T7 Coupled Reticulocyte Lysate System

(Cat.# L4610)

TNT

T3 Coupled Reticulocyte Lysate System

(Cat.# L4950)

TNT

Quick Coupled Transcription/

Translation Systems:

TNT

T7 Quick Coupled Transcription/Translation

System (Cat.# L1170)

TNT

SP6 Quick Coupled Transcription/Translation

System (Cat.# L2080)

TNT

T7 Quick for PCR DNA

(Cat.# L5540)

5’(N)

6–10

TTT

TATTT

AGGTGACACT

TTTAGGTGACACT

TTT

ATA

AGGTGACACTATA

AGGTGACACT

(N)

3–6

CCAC

)

17–22

-3’

SP6 Pr

omoter

Kozak

gion

Sequence-specific

Nucleotide

5’(N)

6–10

TAAT

ACGACTCAC

ATACGACTCAC

TAT

AGGG

TATAGGG

TAT

)

3–6

CCACC

(N)

17–22

-3

’

T7 Pr

omoter

Kozak

gion

Sequence-specific

Nucleotide

Eukar

yotes

SP6

Figure 3.4. Forward primers used to generate PCR fragments for protein expression in TNT

Systems.

33 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

TnT

SP6 High-Yield Wheat Germ Protein

Expression System

In vitro protein synthesis starting from DNA.

Description

The TNT

SP6 High-Yield Wheat Germ Protein Expression System is a convenient, quick, single-tube, coupled transcrip-

tion/translation system designed to express up to 100µg/ml of protein. The

TNT

SP6 High-Yield Wheat Germ Protein

Expression System expresses genes cloned downstream of an SP6 RNA polymerase promoter. This cell-free expression

system is prepared from an optimized wheat germ extract and contains all the components (tRNA, ribosomes, amino

acids, SP6 RNA polymerase, and translation initiation, elongation and termination factors) necessary for protein synthesis

directly from DNA templates. See Table 3.1 for additional applications.

Principle

The TNT

SP6 High-Yield Wheat Germ Protein

Expression System can be used with standard

plasmid DNA or PCR-generated templates containing

the SP6 promoter. However, to achieve optimal

yield, specialized vectors designed for Wheat Germ

Extracts such as pF3A WG (BYDV) Flexi

Vector or

pF3K WG (BYDV) Flexi

Vector are recommended.

DNA templates are directly added to the SP6 High

Yield Master Mix and incubated in a 50µl reaction for

2 hours at 25°C. Expressed proteins can be used

directly or purified for related applications.

Features and Benefits

• Save Time: Generate proteins in two hours,

compared to days when using cell-based (E. coli)

systems.

• Choose Your Format: Use plasmid- or PCR-

generated templates.

• Generate Full-Length Protein: Generate soluble,

full-length protein and avoid problems associated

with E. coli systems.

Ordering Information

TNT

SP6 High-Yield Wheat Germ Protein

Expression System (Cat.# L3260, L3261)

Figure 3.6. Proteins of different size and origin were expressed using TNT

SP6 High-Yield Wheat Germ Protein Expression System in the presence of

FluoroTect

™

tRNA for lysine residue labeling. Samples were separated by

SDS-PAGE and imaged using a fluorescence scanner.

9873TA

TNT

T7 Quick System and

FluoroTect™ tRNA

T7 Quick System and

Transcend™ tRNA

TNT

Wheat Germ System

and FluoroTect™ tRNA

TNT

Wheat Germ System

and Transcend™ tRNA

GFP

eIF4E

Nanos 1

PPP1ca

Luciferase

Minus-DNA

Control

GFP

eIF4E

Nanos 1

PPP1ca

Luciferase

Minus-DNA

Control

GFP

eIF4E

Nanos 1

PPP1ca

Luciferase

Minus-DNA

Control

GFP

eIF4E

Nanos

PPP1ca

Luciferase

Minus-DNA

Contr

250

150

100

102

kDa

102

kDa

250

150

100

kDa

A. B.

C. D.

Transcription and Translation Systems: DNA-based

34 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

TnT

T7 Insect Cell Extract Protein

Expression System

In vitro protein synthesis starting from a DNA template.

Description

The TNT

T7 Insect Cell Extract Protein Expression System is a convenient, quick, single-tube, coupled transcription and

translation system for the cell-free expression of proteins. See Table 3.1 for additional applications.

Principle

The extract is made from the commonly used Spodoptera

frugiperda Sf21 cell line. All components necessary for

transcription and translation are present in the

TNT

Insect Cell Extract (ICE) Master Mix. Proteins are expressed

from genes cloned downstream of the T7 promoter in ICE

vectors such as pF25A or pF25K ICE T7 Flexi

Vector

(Table 9.1). These vectors contain 5´- and 3´-untranslated

(UTR) sequences from the baculovirus polyhedrin gene to

enhance translation efficiency. After addition of the DNA

template, protein synthesis is initiated. The reactions are

incubated at 28–30°C and are complete within 4 hours.

Approximately 15–75µg/ml of functional protein can be

produced using the

TNT

T7 Insect Cell Extract Protein

Expression System.

Features and Benefits

• Obtain Data Faster: Protein is expressed in only

4 hours.

• Achieve High Protein Yields: Express up to

75µg/ml of protein.

• Convenient: Luciferase Control DNA included.

Yield (µg/ml)

Firefly

Luciferase

Renilla

Luciferase

HaloTag

Procaspase-3

Protein

7675MA

Figure 3.7. Typical protein yields using the TNT

T7 Insect Cell Extract

Protein Expression System.

Ordering Information

TNT

T7 Insect Cell Extract Protein

Expression System (Cat.# L1102, L1101)

Transcription and Translation Systems: DNA-based

35 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

E. coli S30 Extract System for Linear Templates

In vitro protein synthesis starting from DNA.

Description

The E. coli S30 Extract System for Linear Templates allows successful transcription/translation of linear DNA templates. You

need only to provide linear DNA containing a prokaryotic E. coli-like promoter (such as lacUV5, tac, λPL (con) and λ-P

). A

ribosome binding site is required to direct the synthesis of proteins in vitro. In vitro-generated RNA from DNA templates

lacking an E. coli promoter may also be used in this system, but protein yields produced from linear DNA templates will be

decreased 1–10%.

Principle

The S30 Extract for Linear Templates is prepared

from an E. coli B strain (SL119), which is deficient in

OmpT endoproteinase, lon protease and exonuclease

V (recBCD). The absence of protease activity results

in greater stability of expressed proteins. The recD

mutation of the SL119 strain produces a more active

S30 Extract for Linear DNA than the previously

described nuclease-deficient, recBC-derived S30

extracts. However, the S30 Extract for Linear Templates

is less active than the S30 Extract System for Circular

DNA. An easy-to-perform, nonradioactive positive

control reaction using the Luciferase Assay Reagent

provided, allows detection of luciferase gene expres-

sion in the E. coli S30 System for linear templates. The

control reaction produces high light output for several

minutes, allowing the researcher to choose from several

detection methods, including simple visual observation

of luminescence.

Features and Benefits

• Flexible: Various templates can be used: DNA fragments,

PCR-synthesized DNA, ligated overlapping oligonucle-

otides, in vitro-generated RNA and prokaryotic RNA.

• Complete: Contains all necessary components for

coupled transcription/translation.

• Optimized: Premix is optimized for each lot of S30

Extract.

• Control DNA: Easy detection of firefly luciferase

expression using (included) Luciferase Assay Reagent.

Ordering Information

E. coli S30 Extract System for Linear

Templates (Cat.# L1030)

Transcription and Translation Systems: DNA-based

36 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

E. coli S30 T7 High-Yield Protein

Expression System

In vitro protein synthesis starting from DNA.

Description

The S30 T7 High-Yield Protein Expression System is an E. coli extract-based protein synthesis system. It simplifies the tran-

scription and translation of DNA sequences cloned in plasmid or lambda vectors containing a T7 promoter, by providing an

extract that contains T7 RNA polymerase for transcription and all necessary components for translation.

Principle

The E. coli S30 T7 High-Yield Protein Expression System

is designed to express up to 500µg/ml of protein in one

hour from plasmid vectors containing a T7 promoter and

a ribosome binding site. The protein expression system

provides an extract that contains T7 RNA polymerase for

transcription and is deficient in OmpT endoproteinase and

lon protease activity. All other necessary components in the

system are optimized for protein expression. This results

in greater stability and enhanced expression of target

proteins. Control DNA expression results in production of

Renilla luciferase, which can be detected by Coomassie

Blue staining following SDS-PAGE or assayed with Renilla

Luciferase Assay System (Cat.# E2810).

Features and Benefits

• Obtain Data Faster: Protein expression in only one hour.

• Achieve High Protein Expression: Express up to

500µg/ml of protein for multiple applications.

• Scalable: Convenient screening protocol for high-

throughput protein expression.

Figure 3.8. Coupled in vitro transcription/translation of circular DNA

templates using the S30 T7 High-Yield Protein Expression System. The

protein-coding sequences cloned into pFN6A (HQ) Flexi

Vector were

expressed as described in the S30 T7 High-Yield Protein Expression

System Technical Manual #TM306, resolved by SDS-PAGE (4–20%

Tris-glycine) and visualized by Coomassie

blue staining (Panel A),

fluorescence scanning (Panel B), or transferred to PVDF membrane,

treated with Streptavidin Alkaline Phosphatase (Cat.# V5591) and stained

with Western Blue

Stabilized Substrate for Alkaline Phosphatase (Cat..#

S3841; Panel C). For each gel: lane 1, no DNA; lane 2, Renilla luciferase;

lane 3, Monster Green

Fluorescent Protein; lane 4, HaloTag

protein; lane

5, α-galactosidase (BCCP = E. coli biotin carboxyl carrier protein).

Ordering Information

S30 T7 High-Yield Protein Expression

System (Cat.# L1110, L1115)

7637T

100

kDa

12345 12345 12345

A. B. C.

BCCP

Transcription and Translation Systems: DNA-based

37 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

37 Discover Reliable Tools for Protein Analysis

Labeling and detection of proteins expressed using cell-free systems is necessary for most applications such as

protein:protein interaction and protein:nucleic acid interaction studies. FluoroTect

™

Detection and Transcend

™

Detection

Systems were developed for non-radioactive protein labeling during cell-free protein synthesis. Both labeling products are

based on the incorporation of labeled lysine residues into the polypeptide chain. The labeled protein products can be easily

detected either by fluorescent imaging after SDS-PAGE or by western blotting using streptavidin conjugates either to horse-

radish peroxidase (Strep-HRP) or Alkaline Phosphatase (Strep-AP).

3.3 Cell-Free Protein Labeling Reagents

OVERVIEW

Figure 3.9. Detection protocols using FluoroTect

™

Green

Lys

tRNA and Transcend

™

tRNA.

Standard Radioisotopic

Incorporation and Detection

Transcend

™

Biotinylated Lysine tRNA

Incorporation and Detection

Translation with

incorporation of

[

S]-met (1 hour)

Translation with

incorporation of

biotinylated lysine (1 hour)

SDS PAGE (1 hour)

Fix gel (30 minutes)

SDS PAGE (1 hour)

Chemiluminescent

detection

Colorimetric

detection

Transfer to PVDF

or nitrocellulose

membrane (1 hour)

Transfer to PVDF

or nitrocellulose

membrane (1 hour)

Block, bind

Strep-AP,

wash (2 hours)

Block, bind

Strep-HRP,

wash (2 hours)

Add

Chemiluminescent

Substrate and

expose to X-ray film

(2−20 minutes)

Add

Western Blue

Substrate to

develop colored

bands (1−10 minutes)

Expose to

X-ray film

(4−10 hours)

Treat with enhancer

(30 minutes)

Dry gel (1 hour)

FluoroTect™ Green

Lys

tRNA

Incorporation and Detection

Translation with

incorporation of

fluorescent lysine

(1 hour)

SDS PAGE (1 hour)

Detection using a

fluoroimaging instrument

(2−5 minutes)

0878MD10_0A

Standard Radioisotopic

Incorporation and Detection

Transcend

™

Biotinylated Lysine tRNA

Incorporation and Detection

Translation with

incorporation of

[

S]-met (1 hour)

Translation with

incorporation of

biotinylated lysine (1 hour)

SDS PAGE (1 hour)

Fix gel (30 minutes)

SDS PAGE (1 hour)

Chemiluminescent

detection

Colorimetric

detection

Transfer to PVDF

or nitrocellulose

membrane (1 hour)

Transfer to PVDF

or nitrocellulose

membrane (1 hour)

Block, bind

Strep-AP,

wash (2 hours)

Block, bind

Strep-HRP,

wash (2 hours)

Add

Chemiluminescent

Substrate and

expose to X-ray film

(2−20 minutes)

Add

Western Blue

Substrate to

develop colored

bands (1−10 minutes)

Expose to

X-ray film

(4−10 hours)

Treat with enhancer

(30 minutes)

Dry gel (1 hour)

FluoroTect™ Green

Lys

tRNA

Incorporation and Detection

Translation with

incorporation of

fluorescent lysine

(1 hour)

SDS PAGE (1 hour)

Detection using a

fluoroimaging instrument

(2−5 minutes)

0878MD10_0A

38 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

Cell-Free Protein Labeling Reagents

FluoroTect

™

Green

Lys

in vitro Translation

Labeling System

Labeling and detection of in vitro synthesized proteins.

Description

The FluoroTect

™

Green

Lys

in vitro Translation Labeling System allows fluorescent labeling and detection of proteins synthesized

in vitro. The system is based on a lysine-charged tRNA, which is labeled at the ε position of the lysine with the fluorophore

BODIPY

-FL. Fluorescent lysine residues will be incorporated into synthesized proteins during in vitro translation reactions,

eliminating the need for radioactivity.

Principle

Detection of the labeled proteins is accomplished in

2–5 minutes directly “in-gel” by use of a fluorescence gel

scanner. This eliminates any requirements for protein gel

manipulation, such as fixing/drying or any safety, regulatory

or waste disposal issues associated with the use of radio-

actively-labeled amino acids. The convenience of “in-gel”

detection also avoids the time-consuming electroblotting

and detection steps of conventional non-isotopic systems.

Features and Benefits

• Fast: Data can be obtained in minutes. No requirement

to transfer, fix or dry gels.

• Nonradioactive: No safety, regulatory or waste

disposal issues associated with radioactivity.

• Flexible: The modified charged tRNA can be used with:

Rabbit Reticulocyte Lysate,

TNT

Coupled Transcription/

Translation System, Wheat Germ Extract and E. coli S30

Extract.

NA

lysine

BODIPY

-FL

(CH

)

0877MD

FluoroTect™ Green

Lys

tRNA

BODIPY

-FL

Cell-free expression system

Lys

Lys Lys

Figure 3.10. Schematic diagram of the incorporation of

FluoroTect

™

Green

Lys

-labeled lysine into nascent protein.

Ordering Information

FluoroTect

™

Green

Lys

in vitro Translation

Labeling System (Cat.# L5001)

39 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

Transcend

™

Nonradioactive Translation

Detection Systems

Labeling and detection of in vitro synthesized proteins.

Description

The Transcend

™

Nonradioactive Translation Detection Systems allow nonradioactive detection of proteins synthesized in

vitro. Using these systems, biotinylated lysine residues are incorporated into nascent proteins during translation, eliminating

the need for labeling with [

S]methionine or other radioactive amino acids

Principle

This biotinylated lysine is added to the translation

reaction as a precharged ε-labeled biotinylated lysine-

tRNA complex (Transcend

™

tRNA) rather than a free

amino acid. After SDS-PAGE and blotting, the bioti-

nylated proteins can be visualized by binding either

Streptavidin-Alkaline Phosphatase (Streptavidin-AP) or

Streptavidin-Horseradish Peroxidase (Streptavidin-HRP),

followed either by colorimetric or chemiluminescent

detection (see Chapter 8). Typically, these methods

can detect 0.5–5ng of protein within 3–4 hours after

gel electrophoresis. This sensitivity is equivalent to

that achieved with [

S]methionine incorporation and

autoradiographic detection 6–12 hours after gel electro-

phoresis.

Features and Benefits

• Sensitive: The biotin tag allows detection of 0.5–5ng

of translated protein.

• Safe: No radioisotope handling, storage or disposal

is required.

• Flexible: Results can be visualized by using

colorimetric or chemiluminescent detection.

Figure 3.11. Schematic diagram of the incorporation of Transcend

™

labeled lysine into nascent protein.

tRNA

lysine spacer arm biotin

Transcend™ Biotinylated tRNA

Translation reaction

Lys

Lys Lys

Biotin

0877MC

Ordering Information

Transcend

™

Colorimetric Translation Detection

System (Cat.# L5070)

Transcend

™

Chemiluminescent Translation

Detection System (Cat.# L5080)

Cell-Free Protein Labeling Reagents

40 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

40 Discover Reliable Tools for Protein Analysis

12291MA

mRN

ranslation

TranslationT

Membrane insertion

otein

Matur

e pr

otein

Signal

peptid

Micr

osomal

membranes

Signal peptide

cleavage

Glycosylation

Microsomal vesicles are used to study cotranslational and initial post-translational processing of proteins. Processing

events such as signal peptide cleavage, membrane insertion, translocation and core glycosylation can be examined by

the translation of the appropriate mRNA in vitro in the presence of these microsomal membranes.

3.4 Membrane Vesicles for Signal Peptide Cleavage

and Core Glycosylation

OVERVIEW

Figure 3.12. Schematic of signal peptide cleavage and introducing core glycosylation by use of canine microsomal membranes in combination

with rabbit reticulocyte lysate cell-free protein expression system.

41 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

Canine Pancreatic Microsomal Membranes

Examination of signal peptide cleavage, membrane insertion, translocation and

core glycosylation of in vitro expressed proteins.

Description

Canine Pancreatic Microsomal Membranes are used to study cotranslational and initial posttranslational processing of

proteins in combination with in vitro expressed protein using Rabbit Reticulocyte Systems (RRLs). Processing events such

as signal peptide cleavage, membrane insertion, translocation and core glycosylation can be examined by the translation of

the appropriate mRNA in vitro in the presence of these microsomal membranes. In addition, processing and glycosylation

events may be studied by transcription/translation of the appropriate DNA in

TNT

RRL Systems.

Principle

Processing and glycosylation events can be studied

with Rabbit Reticulocyte Lysate Cell-free expression

systems. To assure consistent performance with minimal

translational inhibition and background, microsomes

have been isolated free of contaminating membrane

fractions and stripped of endogenous membrane-bound

ribosomes and mRNA. Membrane preparations are

assayed for both signal peptidase and core glycosyl-

ation activities using two different control mRNAs. The

two control mRNAs supplied with this system are the

precursor for β-lactamase (or ampicillin resistance gene

product) from E. coli and the precursor for α-mating

factor (or α-factor gene product) from S. cerevisiae.

Ordering Information

Canine Pancreatic Microsomal Membranes

(Cat.# Y4041)

Membrane Vesicles for Signal Peptide Cleavage and Core Glycosylation

Features and Benefits

• Minimal Translational Inhibition, Minimal

Background: Microsomes are stripped of endogenous

membrane-bound ribosomes and mRNA.

• Compatible: Can be used with

TNT

RRL Systems,

Rabbit Reticulocyte Lysate and Flexi

Lysate.

• Reliable Results: Control mRNAs are supplied.

42 Discover Reliable Tools for Protein Analysis

Cell-Free Protein

Expression Systems

42 Discover Reliable Tools for Protein Analysis

Overview Articles

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reticulocyte lystate expression systems in func-

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Functional Genome/Proteome Analysis

Gene Mutation/Deletion Analysis (e.g., Enzyme

Kinetics)

Park, N., Skern, T. and Gustin, K.E. (2010) Specific

cleavage of the nuclear pore complex protein

Nup62 by a viral protease. J. Biol. Chem. 285(37),

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Protein Domain Mapping

Wong, R.W. and Blobel, G. (2008) Cohesin subunit

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Tando, T. et al. (2010) Requiem protein links RelB/

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Gel Shift EMSA

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Li, M-D. et al. (2012) O-GlcNAc transferase in

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Generation of Protein Arrays

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Protein Evolution/Enzyme Engineering

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Evolution of Antibodies in vitro by Ribosome

Display

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Expression of Difficult-to-Express Proteins

Cell-toxic Proteins, Membrane Protein, Viral

Proteins, Kinases

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Chapter 3 References