An insect cellAn insect cell--free protein synthesis systemfree protein synthesis system

Takashi Suzuki1, Toru Ezure1, Toshihiko Utsumi2 and Eiji Ando1

1 Analytical and Measuring Instruments Division, Shimadzu Corp., Kyoto, Japan2 Appl. Mol. Biosci., Grad. Sch . Med., Yamaguchi University, Yamaguchi, Japan

Amino acids, Energy sources, etc.

Ribosome, Translational elements

Cell Extract

Cell-free (in vitro)Protein synthesis

pTD1

cloning

Expression vector

TranscriptionmRNAmRNA

gene

Buffer

Ex) Expression of -galactosidase

Transdirect insect cell

Sf21 cells

Extraction procedure

Construction of expression vector Reaction composition

Cell-free Protein Synthesis System

T7 Promoter Polh 5'UTR 3'UTRMCS polyA T7

Terminator

pTD1 VectorAmp

T7 Promoter Polh 5'UTR 3'UTRMCS polyA T7

Terminator

pTD1 Vector

T7 Promoter Polh 5'UTR 3'UTRMCS polyA T7

Terminator

pTD1 VectorAmp

Screening of translationalenhancer sequences

Insect Cell Extract Expression vector

Preservation

Baculovirus Polyhedrin 5’UTRs

T7 Promoter 5'UTR 3'UTRMCS polyA T7

Terminator

Ampr

GATATCGAATTCGAGCTCGGTACCCGGGGATCCTCTAGA

Eco RV Kpn ISac I Bam HI Xba I

Sma I

Eco RI

Not IHind III

translational enhancer sequence (46bp) derived from baculovirus (MnNPV)

pTD1 Vector : 3052 bp0

20

40

60

80

100

120

pTD1 -globin%

Transdirect Wheat Germ Rabbit Reticulocyte

Rel

ativ

e pr

oduc

tivity

(%)

Rel

ativ

e pr

oduc

tivity

(%)

Rel

ativ

e pr

oduc

tivity

(%)

0

20

40

60

80

100

120

pTD1 -globin%

Transdirect Wheat Germ Rabbit Reticulocyte

Rel

ativ

e pr

oduc

tivity

(%)

Rel

ativ

e pr

oduc

tivity

(%)

Rel

ativ

e pr

oduc

tivity

(%)

The pTD1 is able to use as a universal vector for

eukaryotic cell-free protein synthesis systems.

The pTD1 is able to use as a universal vector for

eukaryotic cell-free protein synthesis systems.

Transdirect

Maker A

(A) Fluorescent detection

Incorporation of fluorescent labeled lysineduring proteins synthesis reaction.

Rabbit ReticulocyteMaker BTransdirec

tMaker A

(A) Fluorescent detection

Incorporation of fluorescent labeled lysineduring proteins synthesis reaction.

Rabbit ReticulocyteMaker B

B Enzymatic activity

Color measurement of the degraded products of ONPG as a substrate.

TransdirectRabbit Reticulocyte

Maker A Maker B

B Enzymatic activity

Color measurement of the degraded products of ONPG as a substrate.

TransdirectRabbit Reticulocyte

Maker A Maker B

The productivity of Transdirect is applox. 10-fold higher than Rabbit system.The productivity of Transdirect is applox. 10-fold higher than Rabbit system.

Translational enhancer sequence:

Transdirect – baculovirus polyhedrin 5’ UTR

Rabbit Reticulocyte – beta-globin 5’UTR

Fig. 1 Establishment of the insect cell-free protein synthesis system, Transdirect insect cell.

Fig. 2 Synthesis procedure.

Fig. 3 Comparison of protein productivities.

Basic performanceBasic performanceModel protein: b-galactosidase (116kDa)

About 20µg of purified proteins can obtain by affinity purification from 1mL of the reaction mixture.About 20µg of purified proteins can obtain by affinity purification from 1mL of the reaction mixture.

Fig. 4 Affinity purification.

(A) Gal (B) AP (C) tGel(A) Gal (116 kDa)

Strep -galactosidase

(B) AP (47 kDa)

StrepAlkaline phosphatase

(C) tGel (42 kDa)

M 3M 2M R 1

10575

50

160

(kDa)

3035

10575

50

160

(kDa)

30

35

10575

50

160

(kDa)

3035

M 3M 2M R 1

10575

50

160

(kDa)

3035

M R 1

10575

50

160

(kDa)

3035

10575

50

160

(kDa)

30

35

10575

50

160

(kDa)

3035tGelsolin Strep

Strep-tag® : Trp-Ser-His-Pro-Gln-Phe-Glu-LysPurification : Strep-Tactin Superflow (QIAGEN)

Fig. 5 Performance of expression vector, pTD1.

-galactosidase

pTD1 (polyhedrin 5’UTR)

pEU3-NII ( sequence)

pTNT ( -globin leader)

Transdirect Wheat germ

Rabbit reticulocyte lysate

IntroductionIntroductionThe techniques of foreign gene expression

systems are some of the most importanttechnologies in the post-genome era. Cell-free protein synthesis systems are assumed to be powerful tools for such studies, because they are capable of translating exogenous mRNAs with high speed and they have the potential to synthesize any desired proteins, including both native and those that are toxic to cells. In this context, we developed a cell-free protein synthesis system from Spodoptera flugiperda 21 (Sf21) insect cells, and commercialized it as the Transdirect insect cell.In th is poster , we descr ibe i ts basicperformance and some applications of theinsect cell-free system.

TransdirectTransdirect insect cellinsect cellThe Transdirect insect cell is a newly

developed in vitro translation system for mRNA templates, which utilizes an extract from cultured Sf21 insect cells. An expression vector pTD1, which includes a 5'-untranslated region (UTR) sequence from a baculovirus polyhedrin gene as a translational enhancer, was also developed to obtain maximum performance from the insect cell-free protein synthesis system. This combination of insect cell extract and expression vector results in protein productivity of approx. 50 g per mL of the translationreaction mixture (Fig. 1 and 2).

Performance of the kitPerformance of the kitThe expected productivity of target proteins in

the insect cell-free protein synthesis system is approximately 10-fold higher than that in rabbit reticulocyte lysate system (Fig. 3). This is the highest protein productivity yet noted among commercialized cell-free protein synthesis systems based on animal extracts.

Typically, about 20 g of purified proteins were easily obtained by one step of affinity chromato-graphy from 1 mL of translation reaction mixture (Fig. 4).

The pTD1 vector contains all factors involved in mRNA and protein synthesis, including the T7 promoter sequence required for mRNA synthe-sis, the polyhedrin 5'- UTR which enhances the translation reaction, and multiple cloning sites (MCS). The complete DNA sequence of the pTD1 vector is registered in the following DNA Data-bank: DDBJ/GenBank®/EMBL Accession Num-ber AB194742.The translation efficiency of mRNAs trans-

cribed from the pTD1 vector was about 50-fold higher than those of mRNAs without an enhan-cer sequence (data not shown).

Moreover, the pTD1 vector functioned as an effective expression vector not only in the insect cell-free protein synthesis system but also in wheat germ extract and rabbit reticulocyte lysate systems (Fig. 5).

IntroductionIntroductionThe techniques of foreign gene expression

systems are some of the most importanttechnologies in the post-genome era. Cell-free protein synthesis systems are assumed to be powerful tools for such studies, because they are capable of translating exogenous mRNAs with high speed and they have the potential to synthesize any desired proteins, including both native and those that are toxic to cells. In this context, we developed a cell-free protein synthesis system from Spodoptera flugiperda 21 (Sf21) insect cells, and commercialized it as the Transdirect insect cell.In th is poster , we descr ibe i ts basicperformance and some applications of theinsect cell-free system.

TransdirectTransdirect insect cellinsect cellThe Transdirect insect cell is a newly

developed in vitro translation system for mRNA templates, which utilizes an extract from cultured Sf21 insect cells. An expression vector pTD1, which includes a 5'-untranslated region (UTR) sequence from a baculovirus polyhedrin gene as a translational enhancer, was also developed to obtain maximum performance from the insect cell-free protein synthesis system. This combination of insect cell extract and expression vector results in protein productivity of approx. 50 g per mL of the translationreaction mixture (Fig. 1 and 2).

Performance of the kitPerformance of the kitThe expected productivity of target proteins in

the insect cell-free protein synthesis system is approximately 10-fold higher than that in rabbit reticulocyte lysate system (Fig. 3). This is the highest protein productivity yet noted among commercialized cell-free protein synthesis systems based on animal extracts.

Typically, about 20 g of purified proteins were easily obtained by one step of affinity chromato-graphy from 1 mL of translation reaction mixture (Fig. 4).

The pTD1 vector contains all factors involved in mRNA and protein synthesis, including the T7 promoter sequence required for mRNA synthe-sis, the polyhedrin 5'- UTR which enhances the translation reaction, and multiple cloning sites (MCS). The complete DNA sequence of the pTD1 vector is registered in the following DNA Data-bank: DDBJ/GenBank®/EMBL Accession Num-ber AB194742.The translation efficiency of mRNAs trans-

cribed from the pTD1 vector was about 50-fold higher than those of mRNAs without an enhan-cer sequence (data not shown).

Moreover, the pTD1 vector functioned as an effective expression vector not only in the insect cell-free protein synthesis system but also in wheat germ extract and rabbit reticulocyte lysate systems (Fig. 5).

ApplicationsApplications

NH2-Met Gly Xaa Xaa Xaa Ser/Thr Xaa Xaa Xaa1 2 3 4 5 6 7 8

Methionine aminopeptidases

NH2- Gly Xaa Xaa Xaa Ser/Thr Xaa Xaa Xaa

NH- Gly Xaa Xaa Xaa Ser/Thr Xaa Xaa Xaa

N-myristoyl transferasesO

9

ProteinProtein NN--myristoylationmyristoylation

NH2-Met Gly Xaa Xaa Xaa Ser/Thr Xaa Xaa Xaa1 2 3 4 5 6 7 8

Methionine aminopeptidases

NH2- Gly Xaa Xaa Xaa Ser/Thr Xaa Xaa Xaa

NH- Gly Xaa Xaa Xaa Ser/Thr Xaa Xaa Xaa

N-myristoyl transferasesO

9NH2-Met Gly Xaa Xaa Xaa Ser/Thr Xaa Xaa Xaa

1 2 3 4 5 6 7 8

Methionine aminopeptidases

NH2- Gly Xaa Xaa Xaa Ser/Thr Xaa Xaa Xaa

NH- Gly Xaa Xaa Xaa Ser/Thr Xaa Xaa Xaa

N-myristoyl transferasesO

9

ProteinProtein NN--myristoylationmyristoylationProteinProtein NN--myristoylationmyristoylation

tGelsolin strepM-

M

M

G L G L S Y L

L G L S Y LA

Wild-type

G2A mutant

Expression and purification of Expression and purification of tGelsolintGelsolin

Cloning into a pTD1 vector

In vitro transcription

In vitro translation (1 mL scale)with or without the addition of myristoyl-CoA

Affinity purification (Strep-tagsystem, QIAGEN)

SDS-PAGE (result was shown in Fig. 4)

tGelsolin strepM-

M

M

G L G L S Y L

L G L S Y LA

Wild-type

G2A mutant

tGelsolin strepM-

M

M

G L G L S Y L

L G L S Y LA

Wild-type

G2A mutant

Expression and purification of Expression and purification of tGelsolintGelsolin

Cloning into a pTD1 vector

In vitro transcription

In vitro translation (1 mL scale)with or without the addition of myristoyl-CoA

Affinity purification (Strep-tagsystem, QIAGEN)

SDS-PAGE (result was shown in Fig. 4)

PMF analyses of the PMF analyses of the tryptic tryptic digests of digests of tGelsolin tGelsolin proteinsproteinsPMF analyses of the PMF analyses of the tryptic tryptic digests of digests of tGelsolin tGelsolin proteinsproteins

M G L G L S V E R

N-terminal structures Theoretical Observed

1846.9 ND

G L G L S V E R

G L G L S V E R

M A L G L S V E R

A L G L S V E R

A L G L S V E R

1715.9 1716.0

1926.1 1926.2

1860.9 ND

1729.9 1729.8

1940.1 ND

Wild-type

G2A mutant

M G L G L S V E R

N-terminal structures Theoretical Observed

1846.9 ND

G L G L S V E R

G L G L S V E R

M A L G L S V E R

A L G L S V E R

A L G L S V E R

1715.9 1716.0

1926.1 1926.2

1860.9 ND

1729.9 1729.8

1940.1 ND

Wild-type

G2A mutant

Formation of two intramolecule disulfide bonds

Normal DTT free

whole sup. ppt. whole sup. ppt.

Absorbance of hydrolyzed p-Nitrophenyl phosphate was measured at 405nm.

Possible to synthesize active protein which includes S-S bonds

by removing reducing agent (DTT).

Possible to synthesize active protein which includes S-S bonds

by removing reducing agent (DTT).

Normal DTT free- mRNA

ActivityProductivity & Solubility

The synthesized alkaline phosphatases were visualized by incorporating fluorescent labeling lysines.

A DTT free Transdirect is provided as a custom product. Please inquire to t-direct@shimadzu-biotech.jp

A DTT free Transdirect is provided as a custom product. Please inquire to t-direct@shimadzu-biotech.jp

Model protein: E. coli alkaline phosphatase (47kDa)Formation of two intramolecule disulfide bonds

Normal DTT free

whole sup. ppt. whole sup. ppt.

Absorbance of hydrolyzed p-Nitrophenyl phosphate was measured at 405nm.

Possible to synthesize active protein which includes S-S bonds

by removing reducing agent (DTT).

Possible to synthesize active protein which includes S-S bonds

by removing reducing agent (DTT).

Normal DTT free- mRNA

ActivityProductivity & Solubility

The synthesized alkaline phosphatases were visualized by incorporating fluorescent labeling lysines.

A DTT free Transdirect is provided as a custom product. Please inquire to t-direct@shimadzu-biotech.jp

A DTT free Transdirect is provided as a custom product. Please inquire to t-direct@shimadzu-biotech.jp

Model protein: E. coli alkaline phosphatase (47kDa)

Fig. 6 Synthesis of a protein containing disulfide bond .

Protein N-myristoylation.In general, protein N-myristoylation results from the co-translational addition ofmyristic acid to a Gly residue after removal of the initiating Met. The

requirement for Gly is absolute, and Ser or Thr at position 6 is preferred.

MALDI-mass spectra of tryptic digests of the wild-type and G2A mutant.The wild-type mRNA was translated without (upper) or with (middle) the addition of myristoyl-CoA.The G2A mutant was also translated under same conditions (lower). Arrows indicate probable N-terminal prptide peaks.

Fig. 7 Site-directed protein labeling .

Fig. 6 Analysis of protein N-myristoylation . Transdirect has the potential to generate protein N-myristoylation ana N-acetylation.Transdirect has the potential to generate protein N-myristoylation ana N-acetylation.

14070

35

kDa

25

C 1 2 3 4 1 2 3 4FluoroTect TAMRA

50

1 2 3 4 1 2 3 4FluoroTect TAMRA

4base codon (CGGG) Amber codon

14070

35

kDa

25

C 1 2 3 4 1 2 3 4FluoroTect TAMRA

50

1 2 3 4 1 2 3 4FluoroTect TAMRA

4base codon (CGGG) Amber codonLane 2

Lane 3

CAT Strep

Lane 4

CAT StrepMSKQIEVNXSNE-

CAT StrepMX-

In vitro Pin-point Fluorescence Labeling Kit 543 (Olympus)

CloverDirect TAMRA (ProeteinExpress)

4 base codon or amber codon

4 base codon or amber codon

Possible to synthesize site-specific fluorescent labeled protein.Possible to synthesize site-specific fluorescent labeled protein.

C: -galactosidase

1: -mRNA

Protein band

Reduced and alkylation with DTT/iodoacetamide

In gel trypsin digestion

MALDI-TOF MS

MALDI-QIT-TOF MS

Peptide extraction

Protein band

Reduced and alkylation with DTT/iodoacetamide

In gel trypsin digestion

MALDI-TOF MS

MALDI-QIT-TOF MS

Peptide extraction

0

50

100

1700 1750 1800 1850 1900 1950 2000

Wild-type

myrCoA

Wild-type

myrCoA

G2A

myrCoA

m/z 1771.9 (Ac-ALGLS VER)

m/z 1926.2

m/z 1716.0

m/z 1729.8

ApplicationsApplicationsSynthesis of proteins containing disulfide bonds.Escherichia coli alkaline phosphatase (AP)

which contains two disulfide bonds, wasexpressed in a soluble and active form usingthe insect cell-free system under non-reducingconditions. The efficiency of protein synthesisapproached that measured under reducingconditions (norm al kit) (Fig. 6). Humanlysozyme (h-LYZ), which contains four disulfide

bonds, was expressed under non-reducingc o nd i t i on s a f t er ad d i t i on o f r ed uc e d glutathione, oxidized glutathione, and protein disulfide isomerase (data not shown).

Site-directed protein labeling. Four base codon (CGGG) or amber codon (TAG)

was introduced into the N-terminal region of CAT (chloramphenicol acetyltransferase) coding sequence. In the both strategies, position-specific incorporation of fluorescent labeled amino acid was observed using the insect cell-free protein synthesis system.

Analysis of post-translational modifications.Protein N-myristoylation is the important

eukaryote specific lipid modification. To confirmwhether the insect cell-free system has theability to generate N-myristoylation, we chose tGelsolin (truncated human gelsolin) as amyristoylated model protein. Cell-free proteinsynthesis was carried out with or without addition of myristoyl-CoA. The wild-type tGelsolin was found to be N-myristoylated when myristoyl-CoA was added to the translation reaction mixture. Myristoylation did not occur on the G2A mutant, in which the myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiator Met.

ConclusionConclusionThe insect cell-free protein synthesis system

could offer a promising tool to perform gene expression analyses including not only the measurement of enzymatic activity but also investigation of post-translational modifications.

ReferencesReferences1) Ezure, T., et al. (2006) Cell-free protein synthesis system prepared from insect cells byfreeze-thowing. Biotechnol. Prog., 22, 1570-1577.2) Suzuki, T., et al. (2006) Performance of ex-pression vector, pTD1, in insect cell-free trans-lation system. J. Biosci. Bioeng., 102, 69-71.3) Ezure, T., et al. (2007) Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS. Proteomics, 7, 4424-4434.4) Suzuki, T., et al. (2006) N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry. Proteomics, 6, 4486-4495.5) Suzuki, T., et al. (2007) Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry.Proteomics, 7, 1942-1950.

ApplicationsApplicationsSynthesis of proteins containing disulfide bonds.Escherichia coli alkaline phosphatase (AP)

which contains two disulfide bonds, wasexpressed in a soluble and active form usingthe insect cell-free system under non-reducingconditions. The efficiency of protein synthesisapproached that measured under reducingconditions (norm al kit) (Fig. 6). Humanlysozyme (h-LYZ), which contains four disulfide

bonds, was expressed under non-reducingc o nd i t i on s a f t er ad d i t i on o f r ed uc e d glutathione, oxidized glutathione, and protein disulfide isomerase (data not shown).

Site-directed protein labeling. Four base codon (CGGG) or amber codon (TAG)

was introduced into the N-terminal region of CAT (chloramphenicol acetyltransferase) coding sequence. In the both strategies, position-specific incorporation of fluorescent labeled amino acid was observed using the insect cell-free protein synthesis system.

Analysis of post-translational modifications.Protein N-myristoylation is the important

eukaryote specific lipid modification. To confirmwhether the insect cell-free system has theability to generate N-myristoylation, we chose tGelsolin (truncated human gelsolin) as amyristoylated model protein. Cell-free proteinsynthesis was carried out with or without addition of myristoyl-CoA. The wild-type tGelsolin was found to be N-myristoylated when myristoyl-CoA was added to the translation reaction mixture. Myristoylation did not occur on the G2A mutant, in which the myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiator Met.

ConclusionConclusionThe insect cell-free protein synthesis system

could offer a promising tool to perform gene expression analyses including not only the measurement of enzymatic activity but also investigation of post-translational modifications.

ReferencesReferences1) Ezure, T., et al. (2006) Cell-free protein synthesis system prepared from insect cells byfreeze-thowing. Biotechnol. Prog., 22, 1570-1577.2) Suzuki, T., et al. (2006) Performance of ex-pression vector, pTD1, in insect cell-free trans-lation system. J. Biosci. Bioeng., 102, 69-71.3) Ezure, T., et al. (2007) Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS. Proteomics, 7, 4424-4434.4) Suzuki, T., et al. (2006) N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry. Proteomics, 6, 4486-4495.5) Suzuki, T., et al. (2007) Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry.Proteomics, 7, 1942-1950.

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