an insect cell-free protein synthesis system
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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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