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OVERVIEW OF PROTEIN RESEARCH
PRODUCTS & APPLICATIONS . . . . . . . . . . . . . . . . . . 4.2
RECOMBINANT PROTEIN FOLDING
& EXPRESSION
IN VITRO REFOLDING
Refolding CA Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.10
Chaperonin GroE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.11
Protein Disulfide Isomerase (PDI). . . . . . . . . . . . . . . . . . . . . . . . . . . 4.12
Corystein™ (Purothionin) Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . 4.13
N-TERMINAL DEBLOCKING & ANALYSIS
Pfu Pyroglutamate Aminopeptidase . . . . . . . . . . . . . . . . . . . . . . . . 4.14
Pfu Methionine Aminopeptidase . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.15
Pfu Aminopeptidase I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.15
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) . . . . . . . . . . . . 4.16
PROTEIN FRAGMENTATION
Arginylendopeptidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.16
Asparaginylendopeptidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.17
Endoproteinase Asp-N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.17
Acylamino-acud-releasing enzyme. . . . . . . . . . . . . . . . . . . . . . . . . . 4.18
Pfu Protease S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.18
PROTEASE INHIBITION
Calpastatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.18
Table of Contents
Chaperone Plasmid Set. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3
Chaperone Competent Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4
pCold TF DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5
pCold DNA Expression Vectors I-IV . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6
SPP System™ (Single Protein Production System). . . . . . . . . . . . . . 4.8
mRNA Interferase™-MazF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9
PROTEIN SEQUENCING & ANALYSIS
PROTEIN RESEARCH
PROTEIN RESEARCH
C-TERMINAL ISOLATION & ANALYSIS
Carboxypeptidase P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.13
Anhydrotrypsin Agarose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.13
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PROTEIN RESEARCH
Overview of Products and Applications
Takara’s Protein Folding
and Expression Products
Overview
Protein Folding Products
Maximize
solubility
and active
protein
production
Optimize
refolding
conditions of
recombinant
proteins
Chaperonin
GroE
Corystein™
Chaperone
Plasmid
Set
Protein Expression Products
Expression
Vectors
Protein
pCold
Vectors
pCold TF
Vectors
Increase
protein
purity
and
yield
SPP System™
Refolding
CA Kit
(single protein
production)
Chaperonin
GroE
Corystein™
pMazF
Structural
Analysis
(NMR)
Active Soluble Protein
ready for purification
4.2
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3/11/2009 1:25:11 PM
Chaperone Plasmid Set
Cat.# 3340
Application
Set Components
• Correct in vivo Folding of Expressed Recombinant Proteins in E. coli
Plasmid pG-KJE8 (10 ng/µL)
Plasmid pGro7 (10 ng/µL)
Plasmid pKJE7 (10 ng/µL)
Plasmid pGTf2 (10 ng/µL)
Plasmid pTf16 (10 ng/µL)
Description
No. Plasmid
1 pG-KJE8
100 µL
100 µL
100 µL
100 µL
100 µL
Chaperone
Promoter
Inducer
dnaK-dnaJ-grpE
araB
L-Arabinose
groES-groEL
Pzt1
Tetracyclin
Resistant
References
Marker
Cm
2,3
2 pGro7
groES-groEL
araB
L-araBinose
Cm
2
3 pKJE7
dnaK-dnaJ-grpE
araB
L-Arabinose
Cm
2
Compatible E.coli expression systems
4 pG-Tf2
groES-groEL-tig
Pzt1
Tetracyclin
Cm
3
1. These chaperone plasmids carry the pACYC origin of replication and a chloramphenicol resistance gene (Cmr gene); this allows use with standard E. coli
expression systems utilizing ColE1-type plasmids with ampicillin resistance gene
as a marker. This system cannot be used in combination with chloramphenicolresistant E. coli host strains or expression plasmids that carry the chloramphenicol-resistance gene. For example, E. coli BL21(DE3), which is often used with
pET systems etc., is acceptable as a host strain, but E. coli BL21(DE3) pLysS and
BL21(DE3) pLysE, which contains the pLysS or pLysE plasmids which have the
pACYC replication origin and the Cmr gene, cannot be used with this system.
2. This product prescribes a two step method to construct target/chaperone coexpression systems.
The 1st step is to prepare an E. coli host transformed with only the chaperone
plasmid. The 2nd step is to prepare competent cells from this new strain and to
transform this strain with a plasmid expressing the target protein.
5 pTf16
tig
araB
L-Arabinose
Cm
3
Recombinant Protein Folding & Expression
The Chaperone Plasmid Set consists of 5 different plasmids, each of which is
designed to express multiple molecular chaperones that function as a “chaperone
team” to enable protein folding. Co-expression of a target protein with one of these
chaperone teams increases the recovery of soluble proteins. Each plasmid carries
an origin of replication derived from pACYC and a Cmr gene, which allows use with
E. coli expression systems utilizing ColE1-type plasmids containing an ampicillin
resistance gene as a marker. The chaperone genes are situated downstream of an
araB or Pzt-1 (tet) promoter, thus, expression of target proteins and chaperones
can be induced individually if the target gene is placed under the control of other
promoters (e.g. lac). These plasmids also contain the necessary regulator (araC or
tetr) for each promoter.
1 Set
Cmr
Cmr
araC
groEL
araB
dnaK
pG-KJE8
11.1 kb
araC
groES
Pzt1
dnaJ
groES
groEL
Pzt1
araB
Cmr
pKJE7
dnaK
araB
gro ES
pACYC ori
pACYC ori
araC
araB
pACYC ori
grpE
rrnBT1T2
Cmr
pACYC ori
pGro7
5.4 kb
tetR
7.2 kb
araC
dnaJ
tig
gro EL
pG-Tf2
pTf16
8.3 kb
grpE
5 kb
Cmr
tetR
PROTEIN RESEARCH
Chaperone Plasmid Set
tig
pACYC ori
Maps of Takara’s Chaperone Plasmids
Notice
The intellectual property of the plasmids supplied in this product is owned by TAKARA
BIO INC. This product is intended for research purposes only. Use of this product for
commercial purposes such as screening or production requires a separate commercial
contract with TAKARA BIO INC. The sequence information on the plasmids supplied
in this set are not disclosed, and it is prohibited to modify this product or to use this
product for plasmid preparation without prior permission from TAKARA BIO INC.
Possible Model for Chaperone-Assisted Protein Folding in E. coli
Limited Use Label License: [M7][M8]
Takara Bio Europe • www.takara-bio.eu • [email protected]
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PROTEIN RESEARCH
Chaperone Competent Cell
Chaperone Competent Cells BL21 Set
Chaperone Competent Cell pG-KJE8 BL21
Chaperone Competent Cell pGro7/BL21
Chaperone Competent Cell pKJE7/BL21
Chaperone Competent Cell pG-Tf26/BL21
Chaperone Competent Cell pTf16/BL21
TaKaRa Competent Cell BL21
Application
• Convenient and Correct in vivo Folding of Expressed Proteins in E. coli
Recombinant Protein Folding & Expression
Description
Molecular chaperones are extensively-studied proteins involved in the in vivo protein
folding process. E. coli BL21 is an E. coli strain derived from E. coli B which possesses defects in the lon and ompT Outer membrane proteases. E. coli BL21 is commonly used for recombinant protein expression because it generates highly stable
expressed protein.
Takara’s Chaperone Competent Cells are competent Escerichia coli strain BL21 cells
containing one of Takara’s five Chaperone Plasmids.
Takara’s Chaperone Plasmids (pG-KJE8, pGro7, pKJE7, pG-Tf2, pTf16) were developed by the HSP Research Institute, Inc., and are designed for efficient expression
of “chaperone teams”- molecular chaperones which work cooperatively in the cell.
Coexpression of a target protein with one of these “chaperone teams” increases
soluble recovery the target, and may facilitate expression of proteins which are often
unrecoverable by conventional expression methods due to inclusion body formation.
Coexpression of a target protein and chaperone team normally requires three steps:
1. Transformation of host E. coli with a chaperone plasmid; 2. Prepare competent
cells using these transformants and, 3. Transform the cells with a plasmid expressing the target protein. The Chaperone Competent Cells only require one transformation to obtain E. coli coexpressing a target protein and chaperone team. In addition,
TaKaRa’s Competent BL21 cell are available as a control.
These cells can be used for protein expression with Takara’s pCold DNA series. They
cannot be used with the T7 promoter-based expression, such as the pET system,
because the BL21 strain does not express T7 RNA Polymerase.
Cat.# 9120
Cat.# 9121
Cat.# 9122
Cat.# 9123
Cat.# 9124
Cat.# 9125
Cat.# 9126
3 × 6 species × 100 µL
10 × 100 µL
10 × 100 µL
10 × 100 µL
10 × 100 µL
10 × 100 µL
10 × 100 µL
Set Component
Chaperone Competent Cells BL21 Set (Cat.# 9120)
Chaperone Competent Cell pG-KJE8/BL21
Chaperone Competent Cell pGro7/BL21
Chaperone Competent Cell pKJE7/BL21
Chaperone Competent Cell pG-Tf2/BL21
Chaperone Competent Cell pTf16/BL21
TaKaRa Competent Cell BL21
pUC19 DNA (0.1 ng/µL)
SOC medium*
3 × 100 µL
3 × 100 µL
3 × 100 µL
3 × 100 µL
3 × 100 µL
3 × 100 µL
1 × 10 µL
20 × 1 mL
Each Cat.# 9121-9126 contains the following components
Competent Cell
pUC19 DNA (0.1 ng/µl)
SOC medium*
10 × 100 µL
1 × 10 µL
10 × 1 mL
* SOC medium: 2% Tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO4,
10 mM MgCl2, 20 mM Glucose
Storage
–80°C
Related Products
pCold I DNA
pCold II DNA
pCold III DNA
pCold IV DNA
pCold Vector Set
Chaperone Plasmid Set
3361
3362
3363
3364
3360
3340
25 µg
25 µg
25 µg
25 µg
1 Set (ea. 5 µg)
1 Set
References
1) Thomas, J.G., et al. (1997) Appl. Biochem. Biotech.,66, 197-238.
2) Nishihara, K., et al. (1998), Appl. Environ. Microbiol.,64, 1694-1699.
3) Nishihara, K., et al. (2000), Appl. Environ. Microbiol.,66, 884-889.
Limited Use Label License: Cat.# 9120:[M7][M8]; Cat.# 9121, 9122, 9124 and 9125:[M7]
4.4
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3/11/2009 1:25:12 PM
pCold TF DNA
Cat.# 3365
25 µg
Application
Purity
• Vector for Protein Expression using the Cold Shock Promoter (cspA)
• Contains over 70% double-stranded covalently closed circular DNA (RF I)
• Confirmed to maintain cloning sites by dideoxysequencing method
• Shown to cleave at a single site by restriction enzymes Nde I, Sac I, Kpn I, Xho I,
BamH I, EcoR I, Hind III, Sal I, Pst I and Xba I
M
cspA 3’UTR
Multiple cloning site
Factor Xa site
Thrombin site
HRV 3C Protease site
Trigger Factor (TF)
His • Tag
TEE
cspA 5’UTR
lac operator
cspA promoter
IG
13
Recombinant Protein Folding & Expression
pCold TF DNA
Amp
Takara’s pCold TF DNA Vector is a fusion cold shock expression vector that expresses
Trigger Factor (TF) chaperone as a soluble tag. Trigger Factor is a prokaryotic
ribosome-associated chaperone protein (48 kDa) which facilitates co-translational
folding of newly expressed polypeptides. Because of its E. coli origin, TF is highly
expressed in E. coli expression systems. The pCold TF DNA Vector consists of the
cspA promoter plus additional downstream sequences including a 5’ untranslated
region (5’ UTR), a translation enhancing element (TEE), a His-Tag sequence, and a
multicloning site (MCS). A lac operator is inserted downstream of the cspA promoter to ensure strict regulation of expression. Additionally, recognition sites for HRV
3C Protease, Thrombin, and Factor Xa are located between TF-Tag and the Multiple
Cloning Site (MCS) and function to facilitate tag removal from the expressed
fusion protein. Most E. coli strains can serve as expression hosts. The pCold TF DNA
Vector provides cold shock technology for high yield protein expression combined
with Trigger Factor (chaperone) expression to facilitate correct protein folding,
thus enabling efficient soluble protein production for otherwise intractable target
proteins.
(5,769 bp)
lac I
Description
ColE1 ori
pCold TF DNA Vector Map
References
1. Qing, G. et al (2004) Nature Biotechnology 22:877-2004.
2. Gerlined, S., et al (1995) EMBO J. 14:4939-4948.
GenBank Accession No. AB213654
Note: Licensing agreement required.
Limited Use Label Licensing: [L13][L16][M9][M10]
Application: Using the pCold TF DNA
kDa
pCold TF
1 2
pCold
1 2
pCold +
Chaperone
1 2
T7
1 2
Trx
1. Cell extract solution
2. Soluble fraction
target protein
97
66
45
*
*
co-expressed
trigger factor
GST
Nus
1 2 3 1 2 3 1 2 3
pCold pCold I pCold +
Chaperone
TF
1 2 3 1 2 3 1 2 3
kDa
97
66
45
1. Cell extract
solution
2. Soluble
fraction
3. Insoluble
fraction
31
31
22
22
Figure 1. Successful Production of Enzyme Protein A using the pCold TF System.
Expression of this protein, with an estimated molecular weight of 29 kDa, was not seen as
an exact band with either the T7 expression system or even with pCold I (either individual
expression or chaperone co-expression).
However, the expression of the target protein and target plus tag (29 kDa plus 52 kDa) was
observed using pCold TF, and most of the obtained protein was in soluble form. Subsequent
asays confirmed that the expressed enzyme A retains activity even as a fusion protein.
Figure 2. Improved Levels of Soluble Protein B using pCold TF. Expression of soluble
enzyme protein B (M.W: ~63 kDa) was not observed using either pCold DNA I alone or pCold
I co-expressed with chaperone proteins, nor with a T7 expression vector that included other
tags for solubilization (Trx Tag [~12 kDa], Nus Tag [~55 kDa], and GST Tag [~26 kDa]).
However, when the pCold TF DNA Vector was used, the target protein was present at an
expression level much higher than with other systems and tags, and most of the expressed
target protein was observed in the soluble fraction. (Note: The molecular weight of the
target protein is larger than its actual size and varies due to fused expression with different
tags.)
In summary, the pCold TF expression system offers a convenient high yield, high purity
alternative for efficient soluble protein expression of otherwise intractable target proteins.
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04_EU_cat09_PRORES_FINAL_v00.indd 5
PROTEIN RESEARCH
pCold TF DNA
4.5
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PROTEIN RESEARCH
Cold Shock Expression System
pCold DNA
pCold Vector Set
pCold I DNA
pCold II DNA
pCold III DNA
pCold IV DNA
Cat.# 3360
Cat.# 3361
Cat.# 3362
Cat.# 3363
Cat.# 3364
Application
GenBank Accession Nos.
• Protein Expression utilizing the Cold Shock Promoter (cspA).
Description
Recombinant Protein Folding & Expression
1 Set (ea. 5 µg)
25 µg
25 µg
25 µg
25 µg
Takara’s pCold Expression Vectors offer cold shock expression technology
for high purity, high yield protein production.
Takara’s pCold Expression Vectors are four different vectors that utilize the cold
shock Protein A (cspA) promoter for expression of high purity, high yield recombinant protein in E. coli. These vectors selectively induce target protein synthesis at
low temperatures (15°C) where the synthesis of other proteins is suppressed and
protease activity is decreased. This results in high yields of the target protein (60%
of intracellular protein). In addition to the cspA promoter, all four vectors contain a
lac operator (for control of expression), ampicillin resistance gene (ampr), ColE1 origin of replication, M13 IG fragment, and multiple cloning site (MCS). Furthermore,
three of the vectors also contain either a translation enhancing element (TEE), HisTaq sequence, and/or Factor Xa cleavage site. These vectors work equally well for
synthesis of non-labeled and radiolabeled proteins and can be used in conjunction
with Takara’s Chaperone Plasmid Set (3340).
Features
• High Yield Recombinant Protein: Up to 60% of expressed intracellular protein
is target protein.
• Soluble Expression is Increased: Proteins that are insoluble in conventional
expression systems can be expressed in soluble form.
• Wide Range of E.coli Hosts: Compatible with most E.coli strains.
• Can be Combined with Chaperone Plasmids Vectors: When used in conjuction with one of Takara's Chaperone Plasmids, the amount of recoverable
soluble protein can be further increased.
• Radioisotope Labeling: Up to 90% of newly expressed cellular protein is
labeled target protein.
pCold I
pCold II
pCold III
pCold IV
Accession No.
AB186388
AB186389
AB186390
AB186391
Form
10 mM Tris-HCl (pH 8.0), 1 mM EDTA
Chain Length
pCold I DNA :
pCold II DNA :
pCold III DNA :
pCold IV DNA :
4,407 bp
4,392 bp
4,377 bp
4,359 bp
Features of Takara’s pCold Vectors
TEE
His Tag
Ο
Ο
Ο
×
Ο
Ο
×
×
pCold I DNA
pCold II DNA
pCold III DNA
pCold IV DNA
Factor Xa
Cleavage Site
Ο
×
×
×
Purity
• Agaraose Gel Electrophoresis indicates it contains over 70% double-stranded
covalently closed circular DNA (RF I).
• Maintainence of cloning sites was confirmed.
• Single site cleavage by restriction enzymes Nde I, Sac I, Kpn I, Xho I,BamH I, EcoR I,
Hind I, Sal I, Pst I and Xba I was confirmed.
Storage –20°C
Limited Use Label License: Cat.# 3360, 3361, 3362 [L13][L16][M9]; Cat.# 3363, 3364 [L13][M9]
pCold Vector Maps
4.6
04_EU_cat09_PRORES_FINAL_v00.indd 6
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3/11/2009 1:25:17 PM
In the following examples, expression of genes that showed poor expression levels or solubility in the T7 promoter expression system were expressed in the
cold-shock expression system. pCold I DNA was used as an expression vector and BL21 was used as host for expression. Expression from T7 promoter-driven
vectors was conducted using the standard protocol of adding IPTG and culturing at 37°C.
N.C T7 pCold
T7
kDa
T
PROTEIN RESEARCH
Application: Using the pCold Expression Vectors
pCold
T
S
S
kDa
T: Entire protein fraction
S: Soluble fraction
97.4
97.4
← Expression level increased
66.2
66.2
45
45
← Expression enabled
31
21.5
14.4
14.4
CBB staining
CBB staining of the entire protein fraction
Figure 1. Expression of Human Gene A. Human gene A (estimated molecular weight: 31
kDa) was expressed in both the T7 system and the cold-shock expression system. Expression
was not observed in the T7 system, but was observed in the cold-shock expression system.
T7
T
S
pCold
T
S
T7
T: Entire protein fraction
S: Soluble fraction
kDa
Figure 3. Expression of Human Gene C. Comparison of expression of soluble human
gene C protein (estimated molecular weight: 80 kDa) in the cold-shock expression
system versus the T7 system was performed. The expression level of the target protein
in the soluble fraction in the cold-shock expression system was increased dramatically over
expression in the T7 system.
Time after induction
(hours)
0
24
0
pCold™
24
97.4
66.2
Recombinant Protein Folding & Expression
21.5
31
45
31
← Expression level increased
E.coli proteins
other than the
desired protein
are also labeled
21.5
←
Most of the labeled
proteins are the
products of the
desired gene
14.4
CBB staining
Pulse labeling
Figure 2. Expression of Thermophile Gene B. Increased protein expression
and improved solubility were observed when expressing thermophilic gene B (estimated
molecular weight: 30 kDa) in the cold-shock expression system versus the T7 system.
Figure 4. Pulse Labeling of Human Gene D. A comparative study of pulse labeling of
human gene D protein (estimated molecular weight: 12 kDa) using the cold-shock
expression system and the T7 expression system was performed. In the T7 expression
system, other proteins than the target protein were labeled. In the cold-shock expression
system, the vast majority of the expressed labeled protein is protein D, indicating specific
induction of the target.
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04_EU_cat09_PRORES_FINAL_v00.indd 7
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PROTEIN RESEARCH
SPP System™ (Single Protein Production System)
SPP System™ I
SPP System™ II
SPP System™ III
SPP System™ IV
SPP System™ I-IV
Cat.# 3367
Cat.# 3368
Cat.# 3369
Cat.# 3370
Cat.# 3366
Applications
• Convenient for Radioisotope Labeling. Up to 90% of newly expressed
cellular protein is labeled target protein
• NMR Analysis
• Purified Protein Expression
Recombinant Protein Folding and Expression
Description
Takara Bio, in collaboration with Dr. Masayori Inouye at the University of Medicine
and Dentistry of New Jersey, USA, has developed a novel system for generating
large amounts of highly pure, soluble protein. The SPP (Single Protein Production)
System™ utilizes a modified version of Takara’s existing cold shock expression
vectors along with expression of the endoribonuclease mazF to achieve remarkable
protein purity (up to 90% of newly-synthesized protein) and stable isotopic labeling of target proteins. (Figure 1, pg. 4.9).
Principle of the SPP System™
The mazF protein is a sequence-specific endoribonuclease that specifically cleaves
single stranded RNAs at ACA sequences. Expression of mazF virtually eliminates
expression of most cellular mRNAs, while allowing synthesis of a target protein
for up to 72 hours after induction. The SPP System™ consists of two co-expressed
plasmids: an ACA-less pCold expression vector, for expression of an ACA-less
version of the target gene; and a vector carrying the mazF gene, for inducible
expression of the mazF protein. This system suppresses host protein expression
more effectively than the pCold system alone, and results in higher purity and
labeling efficiency for a wider variety of desired proteins. This allows for applications such as protein structural analysis by NMR (nuclear magnetic resonance) or
other technologies.
Features
of the SPP System™
TEE
His-Tag
sequence*
sequence
pCold™ I (SP-4) DNA
pCold™ II (SP-4) DNA
pCold™ III (SP-4) DNA
pCold™ IV (SP-4) DNA
yes
yes
yes
no
yes
yes
no
no
Factor Xa
cleavage site**
yes
no
no
no
* TEE: translation enhancing element. This is a unique sequence that is normally located
14 bases downstream of the initiation codon. It is used to enhance translation initiation
during cold shock.
** Factor Xa cleavage site: This is used in conjunction with the His Tag to increase the
purity of the target protein.
1 kit
1 kit
1 kit
1 kit
1 kit
Kit Components
Cat.# 3367: SPP System™ I
pCold™ I (SP-4) DNA
MazF (mRNA Interferase™) expression plasmid
pMazF DNA
Positive Control pCold™ I (SP-4) envZB DNA
20 µg (0.5µg/µL)
0.5 µg (20ng/µL)
0.2 µg (20ng/µL)
Cat.# 3368: SPP System™ II
pCold™ II (SP-4) DNA
MazF (mRNA Interferase™) expression plasmid
pMazF DNA
Positive Control pCold™ I (SP-4) envZB DNA
20 µg (0.5µg/µL)
0.5 µg (20 ng/µL)
0.2 µg (20 ng/µL)
Cat.# 3369: SPP System™ III
pCold™ III (SP-4) DNA
MazF (mRNA Interferase™) expression plasmid
pMazF DNA
Positive Control pCold™ I (SP-4) envZB DNA
20 µg (0.5µg/µL)
0.5 µg (20 ng/µL)
0.2 µg (20 ng/µL)
Cat.# 3370: SPP System™ IV
pCold™ IV (SP-4) DNA
MazF (mRNA Interferase™) expression plasmid
pMazF DNA
Positive Control pCold™ I (SP-4) envZB DNA
20 µg (0.5µg/µL)
0.5 µg (20 ng/uL)
0.2 µg (20 ng/uL)
Cat.# 3366: SPP System™ I-IV
Cold Shock Expression Vector for SPP System™
pCold™ I (SP-4) DNA, pCold™ II (SP-4) DNA,
pCold™ III (SP-4) DNA, pCold™ SP-4) DNA
MazF (mRNA Interferase™) expression plasmid
pMazF DNA
Positive Control pCold™ I (SP-4) envZB DNA*
20 µg (0.5µg/µL)
0.5 µg (20 ng/µL)
0.2 µg (20 ng/µL)
* Expression plasmid prepared by inserting ORF of E. coli-derived protein envZB without
ACA sequence into pCold™ I (SP-4) DNA. Estimated molecular weight of expressed protein
19.6 kDa.
References
1. Suzuki, M. et al. (2005) Molecular Cell 18:253-261.
2. Zhang, Y. et al. (2004) Journal of Biological Chemistry 280:3143-3150.
3. Zhang, Y. et al. (2003) Molecular Cell 12:913-923.
4. Qing, G. et al. (2004) Nature Biotechnology 22:877-882.
Note: Licensing agreement is required. Please see our website at www.takara-bio.com. The
use of this product is limited for research purposes. It must not be used for clinical purpose or
in vitro diagnosis. Please see our website at www.takara-bio.com.
Limited Use Label License: [L13][L16][L19]
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3/11/2009 1:25:19 PM
Target gene
cspA 3'UTR
multiple cloning site
Factor Xa site
His • Tag
TEE
cspA 5'UTR
lac operator
cspA promoter
(ACA sequence has
already been substituted)
M
IG
13
TM
pCold
MazF cleave ssRNA
at 5'-end of ACA
sequences
ACA
ColE1 ori
ColE1 ori
(including ACA sequence)
cspA 3'UTR
multiple cloning site
TEE
cspA 5'UTR
lac operator
cspA promoter
In cell
Target gene mRNA
M
IG
13
M
ColE1 ori
Figure 1. Principle of SPP System™
(4,359 bp)
r
Expression of host-derived proteins does not happen
due to cleavage on the mRNA by MazF at ACA sites.
pColdTM IV (SP-4) DNA
Amp
(4,377 bp)
r
Amp
Target Protein expressed
lac I
pColdTM III (SP-4) DNA
IG
13
ColE1 ori
Figure 2. Vector Map for SPP System™ Vectors
mRNA Interferase™-MazF
mRNA Interferase™-MazF Cat. #2415A
1,000 U
Application
Purity
• Site-Dependent Cleavage of ssRNA
Other nuclease activities were not detected uder any of the following conditions as
demonstrated by RNA or DNA electrophoresis patterns.
1. After incubation of 40 pmol of ssRNA lacking AC sequences with
20 units of mRNA Interferase™-MazF for 16 hours at 37°C and pH 7.5.
2. After incubation of 40 pmol of dsRNA with 20 units of mRNA Interferase™-MazF
for 16 hours at 37°C and pH 7.5.
3. After incubation of 1 mg of lDNA- Hind III fragments with 20 units of mRNA
Interferase -MazF for 16 hours at 37°C and pH 7.5.
Description
MazF is a toxin protein in the toxin-antitoxin module of E. coli. It possesses
endoribonuclease activity and specifically cleaves single-stranded RNA at the 5’
end of ACA sequences. This enzyme does not cleave double-stranded RNA, doublestranded DNA or single-stranded DNA. This product, mRNA Interferase™-MazF,
is supplied as a fusion protein of E. coli MazF and trigger factor which is an E. coli
chaperone proteins. The enzyme is also supplied with a 5X MazF buffer (200mM
Sodium phosphate, pH7.5, 0.05% Tween-20).
Source
Expressed as a recombinant protein in Escherichia coli
Definition of Activity
One unit is the amount of enzyme that cleaves 1 pmol of standard
substrate (ROX-5’-GATAUACATATCT-eclipse, the underlined regions are
composed of RNA) for 10 min. at 37°C and pH7.5. Buffer composition use for activity definition: 20 mM Sodium phosphate, pH7.5 and 0.05% Tween –20.
Recombinant Protein Folding & Expression
Target gene mRNA is not cleaved
because it does not include
ACA sequence
ACA
Host mRNA
cspA 3'UTR
multiple cloning site
cspA 5'UTR
lac operator
cspA promoter
lac I
Host mRNA
(4,392 bp)
r
r
Target gene mRNA
IG
13
pColdTM II (SP-4) DNA
I (SP-4) DNA
(4,407 bp)
Amp
Amp
In cell
M
lac I
Host genome
cspA 3'UTR
multiple cloning site
His • Tag
TEE
cspA 5'UTR
lac operator
cspA promoter
lac I
MazF gene
pMazF vector
PROTEIN RESEARCH
Outline of SPP System™ Protocol and Vector Map
Note: Licensing agreement required. Please see our website at
www.takara-bio.com. The use of this product is limited for research
purposes. It must not be used for clinical purpose or for in vitro diagnosis.
References
1. Suzuki, M. et al. (2005) Molecular Cell 18, 253-261
2. Zhang, Y. et al. (2004) Journal of Biological Chemistry 280, 3143-3150
3. Zhang, Y. et al. (2003) Molecular Cell 12, 913-923
Limited Use Label License: [L13]
Form
Solution in 20 mM Sodium phosphate (pH6.0), 0.01% Tween-20, 50% glycerol
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04_EU_cat09_PRORES_FINAL_v00.indd 9
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PROTEIN RESEARCH
In vitro Refolding
Refolding CA Kit
Refolding CA Kit
Refolding CA Kit
Cat.# 7350 (small)
Cat.# 7351 (large)
Application
• Refolding of Isolated Inclusion Body Proteins
Description
The Refolding CA Kit uses a novel artificial chaperone technology (licensed from
NFRI, BTRAI, and Ezaki Glico Co, Ltd.) in an easy 2-step procedure for optimizing
the refolding conditions of inclusion body proteins. Optimization results in correct
protein folding and restoration of protein activity.
The small kit is supplied with guanidine hydrochloride and DTT for protein unfolding, four different surfactants that can be added independently to the unfolded
protein solution to provide protection against molecular aggregation, and highly
polymerized cycloamylose (CA), an artificial chaperone, for surfactant removal
and recovery of protein activity. Overnight incubation of the CA-treated protein is
followed by a quick 10-minute centrifugation and collection of the supernatant
containing the refolded protein.
The large kit is used for large scale refolding after the reaction conditions have
been determined using the small Refolding CA kit, and consists of only denaturant
and CA.
25 reactions
1 kit
Kit Components
7350 (Small Kit)
8 M guanidine hydrochloride (GdmCl)
4 M dithiothreitol (DTT)
4 surfactants:
1% Tween 40
1% Tween 60
1% CTAB (cetyltrimethylammoniumbromide)
1% SB3-14 (myristylsulfobetaine)
200 mM DL-cystine
3% CA (highly polymerized cycloamylose)
2 × 1 mL
50 µL
2 × 1 mL
2 × 1 mL
2 × 1 mL
2 × 1 mL
2 × 0.75 mL
7 × 1.6 mL
7351 (Large Kit)
8 M guanidine hydrochloride (GdmCl)
3% CA (highly polymerized cycloamylose)
2 × 10 mL
6 × 20 mL
Related Products
Chaperone Plasmid Set, Cat.# 3340
References
1. Machida, S., et al. (2000) FEBS Lett. 486:131-135.
2. Sundari, C.S., et al. (1999) FEBS Lett. 443:215-219.
3. Daugherty, D.L., et al. (1988) J. Biol. Chem 273:33961-33971.
Limited Use Label License: [L23]
Inclusion body
Suspend in an appropriate buffer.
(recommended concentration : 10 mg protein/ml or less)
Guanidine hydrochlor ide
unfolds inclusion bodies
Suspension of inclusion body
Surfactants prevent
protein aggregation
Add 75 µl of 8 M Guanidine Hydrochloride.
Add 1 µl of 4 M DTT.
Incubate at room temperature for 1 hour.
20 µl of unfolded protein solution
Highly polymer ized CA
removes surfactants and
facilitates protein refolding
Add 1.4 ml of surfactant solution* (+DL-Cystine)*.
*Containing 70 µl of 1% surfactant (and 14 µl of 200 mM
DL-Cystine) and an appropriate buffer.
Incubate at room temperature for 1 hour.
400 µl of reaction mixture.
Biologically active protein in
ther modynamically stable
native confor mation
Add 100 µl of 3% CA.
Incubate overnight at room temperature.
Centrifuge at 15,000 rpm.
Supernatant
Refolded protein solution
Protocol for Refolding CA Kit
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04_EU_cat09_PRORES_FINAL_v00.indd 10
Principle of Refolding CA Kit
Takara Bio Europe • www.takara-bio.eu • [email protected]
3/11/2009 1:25:23 PM
Chaperonin Gro EL
Chaperonin Gro ES
Cat.# 7330
Cat.# 7331
Application
• Refolding of the Denatured Proteins and Restoration of Activity.
5 mg
0.5 mg
PROTEIN RESEARCH
Chaperonin GroE
Description
Chaperonin GroE is a protein complex composed of GroEL (14 subunits, 57 kDa) and
GroES (7 subunits, 10 kDa) and supports proteins in forming their tertiary structure
upon (or immediately after) translation. Data suggests that GroE is essential to
assembly (and presumably reassembly after denaturation) of protein complexes in
vivo. Takara GroE can be used for refolding denatured proteins to recover functional
activity.
Source
In vitro Refolding
Escherichia coli
Form
Lyophilized (contaiing the amount of equivalent to 200 µl of 5mM Tris-HCl buffer,
pH 7.8)
Volume
GroEL: 5 mg protein/vial
GroES: 0.5 mg protein/vial
Storage
–20° C
Purity
≥ 90% on SDS-PAGE
Properties
GroEL:
Molecular weight (one subunit):
Isoelectric point: Optimum pH: Stable pH range: Optimum temperature: Thermal stability : Tolerance to denaturants: 57,000 (amino acid sequence)
60,000 (SDS-PAGE)
4.7
7.0–8.0
6.0–9.0
20–37°C
stable at < 40°C
stable against < 100 mM Guanidine-HCl
Gro ES:
Molecular weight (one subunit): 10,000 (amino acid sequence)
15,000 (SDS-PAGE)
Isoelectric point: 5.0
Optimum pH: 7.0–8.0
Stable pH range: 6.0–9.0
Optimum temperature: 20–37°C
Thermal stability: stable at < 40°C
Tolerance to denaturants: stable against < 100 mM Guanidine-HCl
References
1. Martin, J. et al. (1991) Nature,352, 36-42.
2. Viitanen, P. V. et al. (1991) Biochemistry,30, 9716-9723.
3. Laminet, A. A. et al. (1989) EMBO J.,8, 1469-1477.
4. Mendoza, J. A. et al. (1991) J. Biol. Chem.,266, 13044-13049.
5. Rosenberg, H. F. et al. (1993) J. Biol. Chem.,268, 4499-4503.
6. Brandsch, R. et al. (1992) J. Biol. Chem.,267, 20844-20849.
7. Schmidt, M. et al. (1992) J. Biol. Chem.,267, 16829-16833.
8. Höll-Neugebauer, B. et al. (1991) Biochemistry,30, 11609-11614.
9. Battistoni, A. et al. (1993) FEBS Letters,322, 6-9.
10. Buchner, J. et al. (1991) Biochemistry,30, 1586-1591.
11. Kern, G. et al. (1992) FEBS Letters,305, 203-205.
12. Badcoe, I. G. et al. (1991) Biochemistry,30, 9195-9200.
13. Hartman, D. J. et al. (1993) Proc. Natl. Acad. Sci. USA,90, 2276-2280.
14. Escher, A. et al. (1993) Mol. Genet.,238, 65-73.
15. Kubo, T. et al. (1993) J. Biol. Chem.,268, 19346-19351.
16. Goloubinoff, P. et al. (1989) Nature,342, 884-889.
17. Viitanen, P. V. et al. (1990) Biochemistry,29, 5665-5671.
18. van der Vies, S. M. et al. (1992) Biochemistry,31, 3635-3644.
19. Goloubinoff, P. et al. (1989) Nature,337, 44-47.
20. Grimm, R. et al. (1993) J. Biol. Chem.,268, 5220-5226.
21. Zheng, X. et al. (1993) J. Biol. Chem.,268, 7489-7493.
22. Brunschier, R. et al. (1993) J. Biol. Chem.,268, 2767-2772.
23. Wynn, R. M. et al. (1992) J. Biol. Chem.,267, 12400-12403.
24. Mizobata, T. et al. (1992) J. Biol. Chem.,267, 17773-17779.
25. Fischer, M. T. (1992) Biochemistry,31, 3955-3963.
Takara Bio Europe • www.takara-bio.eu • [email protected]
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PROTEIN RESEARCH
Protein Disulfide-Isomerase (PDI)
Protein Disulfide-Isomerase (PDI)
Cat.# 7318
1 mg
Application
Definition of Activity
• Used for the Refolding of Proteins by Exchange Reactions between
Disulfide Bonds.
One unit of PDI is defined as the amount required to recover one RNase A unit
from reduced bovine RNase A, at 25°C in 15 minutes at pH7.5. One RNase A unit
is defined as the amount required for hydrolysis of cCMP (cytidine 2’ : 3’-cyclic
monophosphate) that results in an increase of 0.001 absorbance unit at 284 nm per
minute, at 25°C in one minute, at pH 7.5.
Description
Protein Disulfide-Isomerase (PDI, Enzyme Code 5.3.4.1) accelerates the exchange
reactions between disulfide bonds in proteins in the presence of appropriate oxidizing or reducing reagents. It is especially useful when working with recombinant
proteins that sometimes lack the tertiary structure of native proteins are not
functional.
In vitro Refolding
PDI Activity
Native Protein
(native disulfides)
SH
SH
Scrambled Protein
(non-native disulfides)
S
S
S
S
PDI
S
S
S
S
Inactive
Activation of Reduced-Denatured or Scrambled Ribonuclease A by PDI
Procedure:
Reduced or scrambled bovine RNase A was incubated with/without PDI at 25°C
under the following reaction conditions. Reactivation of RNase A was assayed in
the presence of cCMP (cytidine 2',3'-cyclic monophosphate) and monitored by the
absorbance at 284 nm.
Reduced Protein
(free sulfhydryls)
SH
SH
Application Example
Reaction Conditions:
Components Substrate Protein
Sodium phosphate buffer, pH 7.5 GSH GSSG PDI final conc.
200 µg/mL
100 mM
2 mM
0.2 mM
20 µg/mL
Results
Active
Reduced RNase
Form
Lyophilized white powder
(Containing the amount equivalent to 200 µL of 50 mM sodium phosphate buffer,
pH 7.5)
Volume
1 mg protein/vial
≥ 300 U/mg
RNase Activity (%)
Bovine liver
RNase Activity (%)
Source
Scrambled RNase
60
60
+ PDI
40
20
+PDI
40
20
–PDI
– PDI
0
0
0
60
30
Time (min)
90
0
30
60
Time (min)
90
Addition of PDI doubled the amount of activity recovered from both the
reduced and scrambled samples.
Storage
–20° C
Purity
Homogeneous on SDS-PAGE
Properties
References
1. Goldberger, R. F., Epstein, C. J. and Anfinsen, C. B. (1964) J. Biol. Chem., 239, 1406.
2. Lambert, N. and Freedman, R. B. (1983) Biochem. J., 213, 225.
3. Hillson, D. A., Lambert, N. and Freedman, R. B. (1984) Methods in Enzymology, 107, 281.
4. Tang, J. G., Wang, C. C. and Tsou, C. L. (1988) Biochem. J., 255, 451.
Molecular weight: 107,000 (homodimer)
Optimum pH: 7.0–9.0
Isoelectric point: 4.2
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Takara Bio Europe • www.takara-bio.eu • [email protected]
3/11/2009 1:25:30 PM
Corystein™ (Purothionin) Reagent Cat.# 7311
Application
Form
• Protein Refolding through Exchange Reactions between Disulfide
Bonds
Lyophilized powder
Description
Homogeneous on SDS-PAGE
Corystein™ (purothionin) is a polypeptide purified from wheat endosperm. This
prod­uct catalyzes the correct formation of disulfide bonds in pro­teins. It can be
used alone or together with thioredoxin to reform disulfide bonds on a variety of
proteins.
Source
Wheat flour
5 mg
Purity
PROTEIN RESEARCH
Corystein™ (Purothionin) Reagent
Properties
Molecular weight: Stable pH range: Isoelectric point: Thermal stability:
5.072 kDa (calculated)
2.0–10.0
10.0
<80°C
Storage
4°C
Carboxypeptidase P
Cat. #7304
100 units
Application
Properties
• C-terminal Sequence Determination
Molecular weight:
51 kDa (gel filtration)
Optimum pH:
3.7 (Cbz-Glu-Try)
4.5* (Cbz-Gly-Pro-Leu-Gly)
5.2 (Cbz-Gly-Lys)
Inhibitors:
**Monoiodoacetic acid (MIA)
diisopropylfluorophosphate (DFP)
p-chloromercuribenzoate (PCMB)
pH stability:
20 min. at pH 3.0–7.0, 30°C
Thermal stability:
10 min. at pH 3.1, 40°C
10 min. at pH 3.7, 50°C
Description
Carboxypeptidase P non-specifically removes amino acids, including proline, from
the carboxy termini of proteins.
Source
Penicillium janthinellum
Definition of Activity
One unit of enzyme activity corresponds to the amount required for hydrolysis 1
µmol of Cbz-Glu-Tyr in 1 minute at pH 3.7, 30°C.
Lyophilized, containing sodium citrate as a stabilizer
* Glycine is rapidly released; a small amount of leu­cine is re­leased at pH 5.2 under
high­er con­cen­tra­tions and prolonged in­cu­ba­tion.
** EDTA and o-phenanthroline do not af­fect ac­tiv­i­ty.
Purity
Storage
Form
4°C (under dry conditions)
No other proteases detected.
In vitro Refolding/C-terminal Isolation and Analysis
Carboxypeptidase P
Anhydrotrypsin Agarose
Anhydrotrypsin Agarose
Cat.# 7302
1 ml wet gel
Application
Volume
• Affinity Chromatography Resin which Selectively adsorbs Peptides containing Arg, Lys or AECys Residues at the C-Terminus.
1.0 ml wet gel/vial
Description
4°C (Stable for at least one year if maintained in 50 mM sodium acetate buffer,
pH 5.0, containing 20 mM CaCl . Do not keep the gel in elution buffer (pH 2.5) for
extended periods.)
Anhydrotrypsin Agarose is an affinity chromatography resin which selectively
adsorbs peptides with Arg, Lys or AECys (S-aminoethyl cysteine) residues at the
C-terminus under weak acidic conditions. It does not bind free amino acids, or
peptides which have these amino acids at positions other than at the C-terminus.
Anhydrotrypsin is a chemical conversion of bovine trypsin, that has a dehydroalanine residue instead of Ser(195) at the catalitic site of trypsin. Anhydrotrypsin
has no detectable catalitic activity but retains a strong affinity for the products of
trypsin digestion. Anhydrotrypsin Agarose consists of anhydrotrypsin immobilized
to an agarose gel by cyanogen bromide.
Storage
2
Binding Capacity
Soybean trypsin inhibitor binding capacity :
Approximately 80 nmol/mL wet gel
Bz-Gly-Arg binding capacity :
Approximately 60 nmol/mL wet gel
Form
Gel suspension in 50 mM sodium acetate buffer, pH 5.0, containing 20 mM CaCl2
and 0.02 % NaN3
Takara Bio Europe • www.takara-bio.eu • [email protected]
04_EU_cat09_PRORES_FINAL_v00.indd 13
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PROTEIN RESEARCH
Pfu Pyroglutamate Aminopeptidase
Pfu Pyroglutamate Aminopeptidase Cat. #7334
Applications
Properties
• Removal of Pyroglutamic Acids from the N-termini of Proteins and
Peptides
• Deblocking of N-terminal Pyroglutamates of Proteins and Peptides for
Sequence Analysis using Edman Degradation
Molecular weight: 24.072 kDa (calculated)
28 kDa (by SDS-PAGE)
Optimum temperature: 95–100°C
Thermal stability: ~90% activity at 75°C + pH 7.0
for 150 min
Optimum pH: 6.0–9.0
Stable pH range:
5.0–9.0
$80% activity in this range 5.0 to 9.0 at 75°C when reacted for 30 min.
Tolerance to denaturants:
#1 M Urea
#1 M Guanidine-HCl
#0.01% SDS
Inhibitors:
PCMB, Hg2+
Description
Pfu Pyroglutamate Aminopeptidase liberates the N-terminal pyroglutamic acid
from proteins and peptides. This enzyme may work well with some intact, nondenatured proteins and, thus, the denaturation step may be unnecessary in these
instances. This product is supplied with 5X Reaction Buffer [250 mM sodium phosphate (pH 7.0), 50 mM DTT, 5 mM EDTA].
N-Terminal Deblocking and Analysis
10 mU
Systematic name
Supplied Buffer (5X)
L-Pyrrolidone carboxyl peptidase. Enzyme code: 3.4.19.3
Volume: 1 ml
Component: 250 mM sodium phosphate buffer (pH7.0) containing
50 mM DTT and 5 mM EDTA
CH2
O=C
|
CH2
|
↓
R1
R2
|
R3
|
|
HN -------CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COCH2
R1
R2
|
O=C
|
|
R3
|
CH2 + NH2-CH-CO-NH-CH-CO-NH-CH-CO|
HN -------CH-COOH
Pfu Pyrogluatmate Aminopeptidase Activity
Comparison of specific activities at various
temperatures (an example)
Enzyme with a specific activity of 5.83 U/mg protein at 37°C showed the activities
of 12.2 U/mg protein or 28.3 U/mg protein at 50°C or 75°C, respectively.
Definition of Activity
One unit of enzyme activity corresponds to the amount required to hydrolyze 1
µmol pyroglutamate p-nitroanilide at 37°C in 1 minute at pH 7.0.
References
1. Shimada, Y. et al. (1989) J. Biochem. 106:383.
2. Hamazume, Y. et al. (1987) J. Biochem. 101:217.
Source
Escherichia coli carrying plasmids encoding the Pyrococcus furiosus pyroglutamate
aminopeptidase gene.
Form
Lyophilized
For your Peptide, Protein
and Antibody Digestion
Volume
10 mU/vial
Purity
$90% on SDS-PAGE. No other proteases detected.
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04_EU_cat09_PRORES_FINAL_v00.indd 14
Takara Bio Europe • www.takara-bio.eu • [email protected]
3/11/2009 1:25:31 PM
Pfu Methionine Aminopeptidase
Cat.# 7335
Properties
• The release of Met Residue from Recombinant Proteins and Peptides
produced by Recombinant Technology.
Molecular weight:
Optimum pH:
Stable pH range:
Optimum temperature:
Thermostability:
Tolerance to denaturants:
Stable against
Activator:
Inhibitor:
Description
Pfu Methionine Aminopeptidase is a thermostable Methionine Aminopeptidase isolated from Pyrococcus furiosus and produced as a recombinant protein. A specifically
liberates the N-terminal methionine residue from proteins and peptides.
Source
Escherichia coli carrying plasmids encoding the Pyrococcus furiosus methionine
aminopeptidase gene.
Form
Solution in 10 mM Tris-HCl, pH 7.5, containing 0.01% Tween 20, and 0.1 mM CoCl2
Volume
32,848 (Mass spectrum analysis)
37,000 (SDS-PAGE)
7.0–8.0
This enzyme is stable at pH 4.1–10.6.
(75°C, 1 hour, 0.5 mM Co2+)
85–95°C
This enzyme is stable for 1 hour at 75°C
(pH 7.2, 0.5 mM Co2+).
≤2 M urea
≤0.2 M guanidine-HCl
≤0.01% SDS
Co2+
EDTA
20 mU at 37°C/vial
Note
Storage
Prior to digestion with this enzyme, it is necessary to denature the protein samples
using a chemical procedure such as carboxymethylation.
–20°C
References
1. Ben -Bassat, A., Bauer, K., et al. (1987) J. Bacteriol.,169, 751.
2. Miller, C. G., Strauch, K. L., et al. (1987) Proc. Natl. Acad. Sci. USA,84, 2718.
3. Ben -Bassat, A., Bauer, K., et al. (1987) Nature,326, 315.
4. Yasueda, H., Nagase, K. et al. (1990) Bio/Technology,8, 1036.
Purity
≥ 95% homogeneous on SDS-PAGE.
No other proteases are detected.
Pfu Aminopeptidase I
Pfu Aminopeptidase I
Cat. # 7336
Application
Purity
• Liberates the N-terminal amino acids up to X-Pro from proteins and
peptides
Homogeneous on SDS-PAGE.
Description
Molecular weight:
Isoelectric point:
Inhibitor:
Optimum pH:
Optimum Temperature:
Thermal Stability:
Pfu Aminopeptidase I is a thermostable exo-type aminopeptidase, isolated from
Pyrococcus furiosus. It is produced as a recombinant protein, which liberates the
N-terminal amino acid from proteins and peptides. This enzyme has a wide range
of substrate specificity, and it does not hydrolyze peptide bonds at the a-amino
residue side of proline (X-Pro). It is significantly activated in the presence of a Co2+
ion.
Source
Escherichia coli carrying plasmids encoding the Pyrococcus furiosus aminopeptidase
I gene.
Form
Lyophilized
0.5 mg
N-Terminal Deblocking and Analysis
Application
20 mU(at 37°C)
PROTEIN RESEARCH
Pfu Methionine Aminopeptidase
Properties
37.483 kDa (calculated) 36–37 kDa (SDS-PAGE)
4.6–4.65
EDTA (Completely inhibited at 0.1 mM)
5.5–8.0 (in the presence of 20 µM Co2+, at 90°C)
80°C (in the presence of 20 µM Co2+,
pH 6.0) 95°C (without Co2+, pH 6.0)
The enzyme retains 65% activity after 4 hrs. at
90°C (pH 8.0, without Co2+).
Activities
• Approximately 84 U/mg protein (5 mM Leucine-p-nitroanilide)
• Approximately 344 U/mg protein (in the presence of 20 µM Co2+, 5 mM Leucinep-nitroanilide)
Definition of Activity
One unit of enzyme activity corresponds to the amount required to hydrolyze 1
µmol of Leucine-p-nitroanilide at 75°C, pH 8.0, in 1 minute.
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PROTEIN RESEARCH
Protein Fragmentation
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP)
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP)
Cat.# 7340
50 µg
Application
Purity
• Liberates the N-Terminal Acyl Blocking Group from Proteins
Homogeneous on PAGE
Description
Properties
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) is a unique exo-type aminopeptidase that liberates blocking groups (formyl, acetyl, and myristyl), and then releases
the subsequent amino acids from proteins and peptides until it reaches the first X-Pro
bond. This enzyme has a wide range of substrate specificities and is used to determine short stretches of amino acid sequence of blocked proteins and peptides whose
sequence can be determined by mass difference in Mass Spectrometry. In addition,
since the amino terminus of Ac-DAP is acetylated, its amino acid sequence cannot
be determined by Edman degradation. Therefore, even if a high E/S ratio is used (for
example, E/S = 0.5–1), the amino acid sequence of the target protein or peptide can
still be determined by Edman Degradation without separation from the enzyme.
Molecular weight:
38,639 (mass spectrometry)
451,000 (by sedimentation equilibrium method)
Activator:
CoCl2
Inhibitor:
Amastatin, EDTA
Optimum pH:
6.5–9.0
Optimum temperature:
85–95°C
Thermal stability: It was confirmed that the enzyme retains 100% activity after 48
hrs at 50°C in the supplied buffer. (pH 8.0, with 0.1 mM Co2+)
Note: Ac-DAP cannot act upon a non-denatured protein; therefore, the protein sample must
be completely denatured by carboxymethylation prior to Ac-DAP digestion.
Source
Yeast carrying the plasmid which contains the gene encoding Pyrococcus furiosus
DAP
Form
Solution in 50 mM N-Ethylmorpholine-AcOH buffer (pH 8.0)
Storage
–20°C
Once thawed, the enzyme solution should be stored at 4°C.
The thawed enzyme and bufffer are stable at least 3 months at 4°C.
Protein Concentration
Supplied Buffer (5×)
Volume :
1 mL
Component :
250 mM N-Ethylmorpholine-AcOH buffer
(pH 8.0) containing 0.5 mM CoCl2
Once thawed, the buffer should be stored at 4°C. Avoid freeze-thaw cycles.
Specific Activity
8-10 units/mg
Definition of Activity
One unit of the enzyme activity corresponds to the amount required to hydrolyze 1
µmol of Leucine-p-nitroanilide at 75°C, pH 8.0, in one minute.
Note
Since this enzyme cannot react with proteins containing the higher order structures,
protein samples should be denatured by chemical procedures such as carboxymethylation. This enzyme cannot act on proteins blotted on PVDF membrane.
Limited Use Label License: [L10]
1 mg/mL
Arginylendopeptidase
Arginylendopeptidase Cat. #7308
0.5 mg
Application
Purity
• Fragmentation of Proteins and Peptides Prior to Structural Analysis
Homogeneous on SDS-PAGE. No other proteases detected.
Description
Properties
Arginylendopeptidase cleaves peptide bonds at the carboxyl side of arginine
residues of pro­teins and pep­tides. Arginylendopeptidase is also known as mouse
submaxillary pro­tease D or as mouse EGF-binding protein C. This enzyme has been
treated with TLCK and TPCK to remove trace trypsin-like and chymotrypsin-like
protease activities. The product is supplied with 5X Reaction Buffer [250 mM sodium phosphate buffer (pH 8.0)].
Note: The enzyme has a weak activity toward -Lys-X- sites, especially when preceded by a basic amino acid residue.
Source
Form
Solution in 5 mM sodium phosphate buffer (pH 7.2) con­tain­ing 50%
glycerol
–20°C
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04_EU_cat09_PRORES_FINAL_v00.indd 16
21.3 kDa (gel filtration)
8.0–9.0
5.65
PMSF, DFP
#2 M Urea
#0.1 M Guanidine-HCl
#0.05% SDS
Definition of Activity
One unit of enzyme activity corresponds to the amount required to produce 1
mmol p-nitroaniline from benzoyl-DL-arginine p-nitroanilide (BAPA) in 1 minute
at 37°C, pH 8.0.
Mouse submaxillary glands
Storage
Molecular weight:
Optimum pH:
Isoelectric point:
Inhibitors: Tolerance to denaturants: References
1. Levy, M. et al. (1970) Meth. Enzymol. 19:672.
2. Schenkein, I. et al. (1977) Arch. Biochem. Biophys. 182:64.
3. Isackson, P. J. et al. (1987) Biochemistry 26:2082.
4. Matsushita, H. et al. (1988) Frontier Forum on Protein Microsequencing.
5. Matsushita, H. et al. (1989) Protein, Nucleic Acid and Enzyme 34:374. (Japanese Journal)
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3/11/2009 1:25:31 PM
Asparaginylendopeptidase
Cat.# 7319
Properties
• Fragmentation of Proteins and Peptides Prior to Structural Analysis
Asparagylendopeptidase specifically cleaves peptide bonds on the carboxyl side
of asparagine residues, including –Asn-Pro– bonds. The substrate specificity of
this enzyme has been validated on many peptides and proteins. The bonds next
to N-terminal or N-second position asparagine residues will not be cleaved with
this enzyme. Bonds adjacent to C-terminal asparagines residues are cleaved. Also,
because asparagine residues bound to sugar chains are not cleaved, this enzyme
can be used to estimate the attachment sites of sugars in glycoproteins of known
sequence. Asparagylendopeptidase is isolated from Jack bean.
Molecular weight: Optimum pH: Stable pH range: Optimum temperature: Thermal stability: Inhibitors: Tolerance to denaturants:
Stable against
Source
Supplied Buffer (5×)
Jack bean
Volume :
Component :
Description
Form
Solution in 20 mM sodium acetate buffer (pH 5.0) containing 50% glycerol, 0.005%
Brij-35, 1 mM DTT, and 1 mM EDTA
Volume
37,000 (SDS-PAGE)
5.5–6.5
4.5–6.5
37–45°C
Stable below 50°C
p-chloromercuribenzoate (PCMB)
N-ethylmaleimide (NEM)
≤ 2 M urea
≤ 0.5 M guanidine-HCl
≤ 0.05% SDS
1 mL
250 mM Sodium acetate, pH 5.0,
50 mM DTT, 5 mM EDTA
Definition of Activity
One unit of enzyme activity corresponds to the amount required to produce 1 µmol
of DNP-Pro-Glu-Ala-Asn from DNP-Pro-Glu-Ala-Asn-NH2 in 1 minute at 37°C, pH5.0.
0.2 mU/vial (5–10 uses for digestion of 2 nmol protein)
Note
Storage
–20°C
It is necessary to denature the protein samples by chemical procedure such as carboxymethylation, prior to digestion with this enzyme.
Purity
Limited Use Label License: [M32]
Protein Fragmentation
Application
0.2 mU
PROTEIN RESEARCH
Asparaginylendopeptidase
No other proteases detected.
Endoproteinase Asp-N
Endoproteinase Asp-N
Cat. # 7329
2 µg
Application
Activity
• Fragmentation of Proteins and Peptides required for Primary Structure
analysis
Approximately 14 U/µg protein
Description
Homogeneous on SDS-PAGE. No other proteases detected.
Endoproteinase Asp-N is a metalloprotease that hydrolyzes peptide bonds on the
amino side of Asp and Cys oxidized to cysteic acid. If cysteine is reduced or alkylated, the enzyme will cleave only the amino side of Asp residues. The enzyme is
supplied with 250 mM sodium phosphate buffer (pH 8.0).
Source
Purity
Properties
Molecular weight: Optimum temperature:
Optimum pH:
Inhibitors: 27 kDa (SDS-PAGE)
37°C
6.0–8.5
2-phenanthroline, EDTA, DTT
Pseudomonas fragi mutant
Definition of Activity
Form
One unit of enzyme activity corresponds to the amount required to increase 0.001
absorbance unit of the peptide soluble in trichloro-acetic acid at 280 nm in 1 minute
at 37°C, pH 8.0 using casein as the substrate.
Lyophilized (containing the equivalent of 50 µL of 10 mM Tris-HCl, pH 7.5)
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PROTEIN RESEARCH
Acylamino-acid-releasing enzyme
Acylamino-acid-releasing enzyme
0.5 U
Application
Supplied Buffer (5×)
• Preparation of Proteins and Peptides for N-Terminal Amino Acid
Sequencing.
Volume :
Component :
Description
Definition of Activity
Aminoacyl-acid-releasing enzyme (AARE) can efficiently liberate the N-terminal
acetylamino acid from N-acetylated peptides of up to about 30 residues in length.
It does not act on intact N-acetylated proteins. Release of the N-acetylamino
acid from proteins is required for Edman-degradation based sequence analysis.
Fragmentation of proteins with residue specific proteases followed by the release of
the N-acetylamino acid from the isolated peptides (using AARE), is required to facilitate N-terminal sequence analysis of proteins.
Systematic Name N-Acylaminoacyl-peptide hydrolase
Protein Fragmentation/Inhibition
Cat.# 7301
Enzyme Code: 3.4.19.1
Source and Form Porcine liver, lyophilized
Volume 0.5 U/vial (Contains about 8 mg of sucrose in each vial)
Storage –20°C (under dry condition) Store reconstituted solution at 4°C.
1 mL
250 mM Sodium phosphate, pH 7.2
One unit of enzyme activity corresponds to the amount required to produce 1 µmol of
L-Ala in 1 minute at 37°C from Ac-Met-Ala at pH 7.2.
Properties
Molecular weight: 360,000 (gel filtration chromatography)
75,000 (SDS-PAGE)
Optimum pH: 7.2–7.6 (Ac-Met-Ala)
Isoelectric point: 4.25
Km value: 0.41 mM (Ac-Met-Ala)
Inhibitors: p-chloromercuribenzonate (PCMB)
diisopropyl fluorophosphate (DFP)
Note: This enzyme cannot react with peptides having Ac-Pro-X-, Ac-Y-Pro-,
Ac-Trp-X-, Ac-Asp-X- or Ac-Glu-X- at their N-termini. This enzyme also cannot react
with peptides having Ac-Met-Asp- at their N-termini.
Purity Homogeneous on SDS-PAGE
Pfu Protease S
Pfu Protease S
Cat. # 7339
500 U
Application
Properties
• Fragmentation of Proteins and Peptides required for Primary Structure
Analysis
Molecular weight:
45 kDa (SDS-PAGE) 42.906 kDa (calculated)
Optimum temperature:
85–95°C
Optimum pH:
6.0–8.0
Inhibitor:
PMSF
Tolerance to denaturants at 95°C pH 7.0:
1% SDS; retains 50% activity after 24 hr.s at 95°C, pH 7.0
6.4 M urea; retains 70% activity after 1 hr. at 95°C, pH 7.0
50% acetonitrile; retains 90% activity after 1 hr at 95°C, pH 7.0
Description
Pfu Protease S is an endo-type serine protease with broad specificity for native and
denatured proteins. Cleavage occurs mainly on the carboxy side of peptide bonds of
hydrophobic amino acid residues.
Source
Bacillus species carrying a plasmid that encodes the Pyrococcus furiosus protease
gene
Form Solution in 25 mM Tris-HCl (pH 7.6) containing 40% ethanol
Purity Homogeneous on SDS-PAGE. No other proteases detected.
Definition of Activity
One unit of enzyme activity corresponds to the amount required to hydrolyze 1
mmol of N-succinyl Ala-Ala-Pro-Phe p-nitroanilide in 1 minute at 95°C, pH 7.0.
Limited Use Label License: [L10][M36]
Calpastatin
Calpastatin
Cat. #7316
3 mg
Application
Purity Homogeneous on SDS-polyacrylamide gel elec­tro­phore­sis.
• Calpain Protease Inhibitor
Properties
Description
Molecular weight:
Peptide length:
Calpastatin is an endogenous protease in­hib­i­tor that acts specifically on calpain (a
calcium-dependent cysteine pro­tease). It consists of four repetitive sequences of 120
to 140 amino acid residues (domains I, II, and IV), and an N-ter­mi­nal non-homologous
sequence (L). The prod­uct consists of highly purified recombinant human calpastatin
domain I .
Form Lyophilized white powder
Source Recombinant Human Calpastatin Domain I
Storage –20°C
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14 kDa
137 a.a.
Typical Activity
50 nM of calpastatin domain I completely inhibits the activity of 7.5 µg/mL calpain I.
15 nM of calpastatin domain I inhibits 50% of the activity of 7.5 µg/mL calpain I.
References
1. Uemori, T. et al. (1990) Biochem. Biophys. Res. Comm. 166:1485.
2. Asada, K. et al. (1989) J. Enz. In­hi­b. 3:49.
3. Kanaki, R. and Murachi, T. (1987) Protein, Nucleic Acid and
En­zyme 32:116 (Japanese Journal).
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3/11/2009 1:25:32 PM