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Protein Expression, Folding and Purification
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P R O T E I N S T U D I E S S E L E C T I O N G U I D E
3
T A B L E O F C O N T E N T
EXPRESSION SYSTEMS
Bacterial Expression & Purification—pET Express & Purify Kits ........................................................................ 4
HAT Protein Expression & Purification System .................................................................................................... 4
B. subtilis Secretory Protein Expression System ................................................................................................. 5
Brevibacillus Expression System II ........................................................................................................................ 6
BacPAK™ Baculovirus Expression System ........................................................................................................... 8
In-Fusion® Ready BacPAK™ Vector Set ................................................................................................................. 8
Baculovirus Accessory Products ............................................................................................................................ 8
BacPAK™ Baculovirus Rapid Titer Kit ............................................................................................................... 8
BacPAK™ qPCR Titration Kit .............................................................................................................................. 9
INCREASED PROTEIN YIELD AND PURITY
pCold Expression Vectors ..................................................................................................................................... 10
pCold TF Vectors .....................................................................................................................................................11
pCold ProS2 DNA ....................................................................................................................................................11
mRNA Interferase™- MazF Enzyme ......................................................................................................................11
SPP System ............................................................................................................................................................. 12
CELL FREE PROTEIN EXPRESSION
Human Cell-Free Expression System ................................................................................................................... 13
CLONING METHOD FOR PROTEIN EXPRESSION
Optimal PCR-cloning method for protein expression ........................................................................................ 15
PCR enzymes perfect for In-Fusion® cloning! ..................................................................................................... 15
EXPRESSION VECTORS OVERVIEW
Overview table ....................................................................................................................................................... 16
FOLDING
Chaperone Plasmid Set ......................................................................................................................................... 18
Chaperonin GroE .................................................................................................................................................... 19
Corystein™ (Purothionin) Reagent ....................................................................................................................... 19
Refolding CA Kit ..................................................................................................................................................... 19
HIS TAGGED PROTEIN PURIFICATION
Talon® Co Resin-Highest Purity ............................................................................................................................ 20
His60 Ni SuperflowTM Resin-Highest Capacity .................................................................................................... 20
OTHER PROTEIN PURIFICATION
DYKDDDDK (FLAG Epitope) Immunoprecipitation and Detection .................................................................... 24
GST-Tag Purification Resins .................................................................................................................................. 24
c-Myc and HA-tagged proteins Detection and purification ............................................................................... 25
Other purification products ................................................................................................................................... 25
PROTEIN SEQUENCING AND ANALYSIS
Takara’s Protein Sequencing and Analysis Products ......................................................................................... 27
4
Bacterial Expression & Purification—pET Express & Purify Kits• Powerful—Higher inducible protein expression levels & tighter control of your target gene• Fast—E. coli BL21(DE3)-based system• Versatile—Includes N- & C-terminal 6xHN-tagged vectors• Convenient—Choice of fast, simple In-Fusion PCR cloning or traditional T4 DNA ligase cloning• Complete—Purify your expressed protein with His60 Nickel resin or TALON® Cobalt resinThe pET expression system is the most commonly used bacterial system for the over-expression of genes. It is a complete system with 6xHN-tagged vectors together with IMAC-based purification. Our pET system utilizes two levels of regulation to provide the highest level of protein expression and the tightest control over basal expression—achieved via the presence of lac operator sites in two different promoters.
• The first level of regulation is provided by the pET6xHN series of vectors. The gene of interest is cloned downstream of a strong T7 lac hybrid promoter. T7 RNA polymerase is extremely selective in binding to this hybrid promoter, thereby utilizing most of the cell’s resources to express this gene.
• The second level of regulation occurs in the host cell. The T7 RNA polymerase gene is integrated into the host genome under the control of the lac UV5 promoter, which also contains a lac operator. This enables expression of T7 RNA polymerase to be controlled by the lacI genes present in both the host genome and the pET6xHN vectors, which encode lac repressor.
The pET Express & Purify System Vectors—pET6xHN
Clontech’s pET Express and Purify kits contain pET vectors (the pET6XHN series of vectors) which encode N- or C-terminal 6xHN fusion tags.
These are available in a choice of cloning formats (easy In-Fusion cloning or traditional restriction enzyme cloning) for maximum flexibility tailored to your expression needs.
pET Express & Purify Kits—a Complete Expression & Purification System
The pET Express & Purify kits supply a choice of IMAC resins and buffers to purify expressed his-tagged proteins. You can choose His60 Ni nickel-based resin for high binding capacity or HisTALON cobalt-based resin for high purity. The kits are supplied with prepacked gravity columns filled with either resin and provide all of the buffers necessary to perform the protein extraction and purification.
The pET Express & Purify Kit protocol.
Product InformationBrand Product Size Cat.No
Clontech pET Express & Purify Kit—His60 20 Purifications 631431
Clontech pET Express & Purify Kit—His60 (In-Fusion Ready) 20 Purifications 631428
Clontech pET Express & Purify Kit—HisTALON 20 Purifications 631430
Clontech pET Express & Purify Kit—HisTALON (In-Fusion Ready) 20 Purifications 631429
Clontech HAT Protein Expression & Purification System Each 631205
Clontech pHAT20 Vector 20 ug 631202
E X P R E S S I O N S Y S T E M S
The HAT System provides an easy and efficient way to express and purify proteins in bacteria. The HAT (histidine affinity tag) epitope tag consists of a naturally-occurring sequence of non-adjacent histidine residues, and has a lower overall charge than tags with consecutive histidine residues.
HAT Protein Expression & Purification SystemThe three included pHAT Vectors—pHAT10/11/12 are the same construct with an enterokinase (EK) cleavage site but have MCS in each of the three reading frames . TALON resin and buffers are included in the kit for the purification of HAT-tagged proteins. The pHAT20 Vector, available separately, provides all the advantages of the pHAT10/11/12 vectors along with the convenience of additional cloning sites.
5
E X P R E S S I O N S Y S T E M S
B. subtilis Secretory Protein Expression System
Flowchart of the experimental procedure for the B. subtilis Secretory Protein Expression System.
aprEpromoter
Ampr
pBE-S DNA(5,938 bp)
aprE SP
multi cloning site (MCS)
ColE1 ori
pUB ori
Kanr
Mlu I
Eco52 I
His-Tag
173 different types of SP DNA are inserted into this region in place of the aprE SP
Vector map for pBE-S DNA, a B. subtilis/E. coli shuttle vector used with the B. subtilis Secretory Protein Expression System.
Features
• Includes pBE-S DNA, an E. coli/B. subtilis shuttle vector with B. subtilis-derived subtilisin (aprE) promoter, secretory signal peptide (aprE SP), Multiple Cloning Site, and 3’ (C-terminal) His-tag sequence
• Supplied with SP DNA Mixture, a library of DNA sequences encoding 173 unique secretory signal peptides that can be inserted upstream of your target gene
• Fully compatible with In-Fusion cloning kits and systems to allow rapid and easy construct generation
• Includes B. subtilis strain RIK1285
Applications
• Expression of soluble, recombinant protein secreted directly into the culture media
• Protein expression in a host amenable to medium- and large-scale fermentation in addition to small-scale culturing
• Expression of proteins with complex structure, such as proteins with disulfide (S-S) bonds
• Generation of target protein in a host that is considered to be Generally Regarded As Safe (GRAS) by the U.S. Food and Drug Administration
• Useful for producing easily purified recombinant protein – with proper in-frame cloning, a C-terminal His-tag can aid purification from culture media
Description
Bacillus subtilis has become an increasingly popular host for recombinant protein expression. With its ability to secrete protein directly into culture media, amenability to medium- and large-scale fermentation, lack of codon bias, and designation by the U.S. Food and Drug Administration as an organism that is Generally Regarded As Safe (GRAS), it’s no wonder that the majority of industrially-produced enzymes are expressed in Bacillus species such as B. subtilis. Optimization
Related In-Fusion Cloning ProductsFor rapid and easy cloning, use the In-Fusion HD Cloning System: see page 15
Results of measuring β-glycosidase activity of 470 clones of an expression library with different signal peptide sequences.Clones showing activity levels of varying strengths were observed. The arrowheads indicate the expression level observed with the aprE signal peptide.
A405
321 331 341 351 361 371 381 391 401 411 421 431 441 451 46100.511.522.53
A405
161 171 181 191 201 211 221 231 241 251 261 271 281 291 301 31100.511.522.53
A405
1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 15100.511.522.53
◄
◄
◄
of secretion, however, can be necessary to achieve highest yields. To address this, the B. subtilis Secretory Protein Expression System from Takara Bio allows rapid development of a library of B. subtilis clones, each bearing a pBE-S construct in which the ORF for your protein of interest is fused with sequences for 173 unique signal peptides. Perform a downstream assay to identify and select clones which secrete the highest amount of functional protein into the culture media, and you can quickly identify the signal peptide that results in efficient expression of your desired secreted protein.
Product InformationBrand Product Size Cat.No
Takara B. subtilis Secretory Protein Expression System
10 rxns 3380
Screening of target protein expression or activity
Insertion of a target gene into the MCS of pBE-S DNA
Linearization of the plasmid by cleaving it with Mlu I and Eco521
Insertion of SP DNA mixture by In-FusionTM reaction
Transformation into E.coli HST08
Preparation of plasmid library
Transformation into B.subtilis (e.g., strain RIK1285)
6
Brevibacillus Expression System II
Features
• Efficient production of secreted or intracellular target proteins• Produces negligible amounts of extracellular protease – products remain intact in culture medium• Unlike E. coli, produces no endotoxins• Proteins are produced in active form• Easy to culture, handle, and sterilize
Description
Brevibacillus choshinensis is a gram-positive bacterium with exceptional capacity for heterologous protein expression.
The Brevibacillus Expression System II enables highly efficient production of target protein in a secreted form. This system allows high yield of active proteins and is well-suited for expression of eukaryotic proteins. The Brevibacillus system is nearly free of proteases, which facilitates production of intact protein products.
Examples of successfully expressed proteins can be seen in Table 1. This includes expression of enzymes, antigens, and cytokines. Each protein was produced at a very high level of expression and confirmed to have native biological activity. In addition, proteins from taxonomically distant organisms were successfully produced, such as eubacteria, archaebacteria, eukaryotes, and viruses.
Table 1: Example of Proteins Expressed using the Brevibacillus Expression System.
Proteins Origins Production( /L )Enzymes
α-amylase B. licheniformis 3.7
Sphingomyelinase B. cereus 3
Xylanase B. halodurans 0.2
CGTase B. macerans 1.5
Chitosanase B. circulans 1.4
Hyper thermo-stable protease A. pernix 0.1
Hyper thermo-stable nuclease P. horikoshii 0.7
PDI human 1
Antigens
Surface antigen E. rhusiopathiae 0.9
Surface antigen T. pallidum 0.8
Cytokin s
EGF human 1.5
IL-2 human 0.6
NGF mouse 0.2
IFN- γ chicken 0.5
TNF-α bovine 0.4
GM-CSF bovine 0.2
GH flounder 0.2
Proteins Origins Production( g/L
α-amylase B. licheniformis 3.7
Sphingomyelinase B. cereus 3.0
Xylanase B. halodurans 0.2
CGTase B. macerans 1.5
Chitosanase B. circulans 1.4
Hyper thermo-stable protease A. pernix 0.1
Hyper thermo-stable nuclease P. horikoshii 0.7
PDI human 1.0
Surface antigen E. rhusiopathiae 0.9
Surface antigen T. pallidum 0.8
e
EGF human 1.5
IL-2 human 0.6
NGF mouse 0.2
IFN- γ chicken 0.5
TNF-α bovine 0.4
GM-CSF bovine 0.2
GH flounder 0.2
E X P R E S S I O N S Y S T E M S
The Brevibacillus system facilitates disulfide bond formation (commonly required in proteins of eukaryotic origin). In addition, B. choshinensis serves as an excellent host for intracellular protein production, frequently producing intracellular proteins in a soluble form in the cytoplasm without forming inclusion bodies. The Brevibacillus system often works better than E. coli for the expression of particular targets.
Utilizing His-tag containing vectors (pNC-HisE, pNC-HisF, pNC-HisT, pNI-His) allows effective purification of the expressed target protein. Tags can be removed by protease treatment following purification.
Product InformationBrand Product Size Cat.No
Takara Brevibacillus Expression System II 1 Kit HB200
Takara Brevibacillus choshinensis Competent Cells 100 µL x 10 HB116
Takara pNC-HisE DNA 10 µg HB123
Takara pNC-HisF DNA 10 µg HB122
Takara pNC-HisT DNA 10 µg HB121
Takara pNCMO2 DNA 10 µg HB112
Takara pNY326-BLA DNA 1 µg HB114
Takara pNI DNA 10 µg HB131
Takara pNI-His DNA 10 µg HB132
Takara pNY326 DNA 10 µg HB111
7
E X P R E S S I O N S Y S T E M S
Brand Vector Name Vector TypeExpression
VectorLac
OperatorHis-Tag
Sec Signal
PeptideConstruct in
Protease cleavage site
X-terminator
Takara pNC-HisE (5,263 bp) Shuttle Vector Secretory Yes Yes Yes E. coli Enterokinase No
Takara pNC-HisT (5,260 bp) Shuttle Vector Secretory Yes Yes Yes E. coli Thrombin No
Takara pNC-HisF (5,260 bp) Shuttle Vector Secretory Yes Yes Yes E. coli Factor Xa No
Takara pNI-His DNA (5,079 bp) Shuttle Vector Intracellular Yes Yes No E. coli Enterokinase No
Takara pNI DNA (5,055 bp) Shuttle Vector Intracellular Yes No No E. coli No No
Takara pNY326 (3.4 Kb) Expression Secretory No No Yes Brevibacillus No Yes
Takara pNCMO2 (5.2 kb) Shuttle Vector Secretory No No Yes E. coli No Yes
Takara pNY326-BLA Positive Control
Secretory Includes a gene encoding Bacillus licheniformis a-amylase (55 kDa)
Vectors for the Brevibacillus System
Choosing a Brevibacillus Vector?
All shuttle vectors between B. choshinensis and E. coli contain the P2 promoter, which is one of the five promoters that control transcription of the cell wall protein gene (HWP). This promoter functions only in B. choshinensis and not in E. coli, thereby ensuring robust protein production only in B. choshinensis.
Nmrrep
ori –P5 promoter
sec signal peptide
multiple cloning site
X-terminator
ori +
pNY326(3.4 kp)
Amp r
ColE1 ori
Nm r
rep
ori +
ori –
pNCMO2 (5.2 kb)
P2 promoterLac operator
sec signal peptide
His-TagMCS
ori +
rep
NmR
AmpR
ColE1 ori
ori –
pNC-HisT(5,260 bp)
P2 promoterLac operator
sec signal peptide
His-TagMCS
ori+
rep
NmR
AmpR
ColE1 ori
ori-
pNC-HisF(5,260 bp)
P2 promoterLac operator
sec signal peptide
His-TagMCS
ori+
rep
NmR
AmpR
ColE1 ori
ori-
pNC-HisE(5,263 bp)
P2 promoter Lac operator
multi cloning site
ori +
rep
Nmr
Ampr
ColE1 ori
ori –
pNI-His DNA(5,079 bp)
His-TagP2 promoter
Lac operator
multi cloning site
ori +
rep
Nmr
Ampr
ColE1 ori
ori –
pNI DNA(5,055 bp)
The pNY326 vector* is maintained more stably than pNC or pNI vectors in the host cells due to much weaker promoter activity and smaller size (3.4 kb). The host strains containing the vector can be repeatedly subcultured, may be used for scaled-up production, and will continue to stably produce protein. The pNY326 vector must be constructed by a one-step method using B. choshinensis. Brevibacillus choshinensis Competent Cells are used as the transformation host.*Can only be maintained in B. choshinensis.
Application note
High-level Secretion of Recombinant Protein using the Brevibacillus Expression SystemBy Michikazu Tanio and Toshiyuki Kohno, Mitsubishi Kagaku Institute of Life Sciences (MITILS), Tokyo, Japanwww.takara-bio.eu/appnote-brevi.pdf
8
BacPAK™ Baculovirus Expression System
E X P R E S S I O N S Y S T E M S
The BacPAK Baculovirus Expression System expresses recombinant proteins at extremely high levels (1 to 500 mg of protein per liter of culture) in insect host cells (1, 2). The BacPAK System offers three major advantages:
• High yield of recombinant protein. The insect host cells produce large amounts of your target protein.
• Greater similarity to naturally occurring proteins. The expressed recombinant protein is usually similar in structure, biological activity, and immunological reactivity to the naturally occurring protein because insect host cells provide post-translational processing similar to that of mammalian cells.
• High recombination efficiency. More than 90% of the viruses produced by the transfected cells carry the target protein. The specially designed BacPAK6 Viral DNA forces recombination between the virus and transfer vector, resulting in high recombination efficiency.
In-Fusion® Ready BacPAK™ Vector Set• In-Fusion technology greatly simplifies cloning• Add N- or C-terminal polyhistidine tags• Obtain high protein purity using TALON® resins Baculovirus expression offers a significant advantage over bacterial expression for generating large amounts of a recombinant protein, since the post-translational processing and folding of recombinant proteins produced in insect cells closely resembles mammalian processing and the yields of functional protein are often much greater. The In-Fusion Ready BacPAK Vector Set allows proteins to be quickly and easily overexpressed in insect cells using In-Fusion cloning technology, and efficiently purified using TALON Resin.
In-Fusion Cloning Simplifies ExpressionIn-Fusion cloning speeds up the preparation of baculoviral transfer vectors. It is simple, fast, accurate, directional, and allows PCR products up to 15 kb in length to be directly cloned without digestion or blunt-end polishing.
The In-Fusion Ready BacPAK Vector Set and Baculovirus Expression System. A PCR fragment containing your gene of interest is simultaneously and directly cloned into the In-Fusion Ready BacPAK Vector pair to generate N- and C-terminal 6xHN-tagged constructs.
PCR product of yourgene of interest
with In-Fusion ends
In-Fusion cloningand transformation
into E. coli
Miniprep and sequence clones
Sf9 or Sf21 insect cells
Harvest culture supernatant and amplify virusConduct small scale analysis
Large scale infection and expressionof recombinant protein
Purify by using TALON resin
Cotransfect pBacPAK plasmid and BacPAK6linear DNA into Sf9 or Sf21 insect cells
6xHN
AcMNPVSequence
LinearBacPAK6
DNA
pBacPAKNterm-6xHN
Poly AStop
Stop
AcMNPVSequence
6xHNATG
AcMNPVSequence
LinearBacPAK6DNA
pBacPAKCterm-6xHN
Poly AAcMNPV
Sequence
Ppolyhedrin
Ppolyhedrin
ATG
The In-Fusion Ready BacPAK vectors are prelinearized and require no restriction enzyme digestion, phosphatase treatment, or gel purification prior to cloning (1).References1. In-Fusion Ready BacPAK Vector Set (2006) Clontechniques XXI(2):16-17.
BacPAK MethodThe target gene is inserted into a shuttle vector, which is co-transfected into insect host cells with the linearized BacPAK6 Viral DNA. The BacPAK6 DNA is missing an essential portion of the baculovirus genome. When the DNA recombines with the vector, the essential element is restored and the target gene is transferred to the baculovirus genome. Following recombination, a few viral plaques are picked and purified, and the recombinant phenotype is verified. The newly isolated recombinant virus can then be amplified and used to infect insect cell cultures to produce large amounts of the desired protein.References1. Kitts, P. A. & Possee, R. D. (1993) Biotechniques 15(5):810–817.2. Kitts, P. A. et al. (1990) Nucleic Acids Res. 18:5667–5672.
Baculovirus Accessory ProductsBacPAK™ Baculovirus Rapid Titer Kit• Saves time by shortening baculovirus expression experiments by up to six days • Eliminates troublesome plaque assays • Compatible with all commonly available (AcMNPV-based baculovirus expression systems The BacPAK Baculovirus Rapid Titer Kit provides a fast and simple method for determining titers of baculovirus stocks, typically the most time-consuming part of baculovirus expression protocols. The kit uses a standard immunological assay to accurately determine baculovirus titers within 48 hours, whereas other methods, such as plaque and end-point dilution assays, require 4–8 days.
In the BacPAK Baculovirus Expression Systems, infected cells express viral antigens long before plaques are formed. Therefore, the Bac-PAK Rapid Titer assay allows titer determination after a much shorter incubation period than traditional plaque assays (1). Furthermore, the titers obtained with the Rapid Titer assay are comparable to those obtained with other methods. This kit is suitable for use with any virus stock with a titer of more than 104 pfu/ml and is compatible with all commonly available (AcMNPV-based) baculovirus expression systems.References1. Wolkman, L. E. & Goldsmith, P. A. (1982) Appl. Envir. Microbiol. 44
(1):227-233.
9
E X P R E S S I O N S Y S T E M S
BacPAK™ qPCR Titration Kit
• Determine viral titers in 4 hours with this rapid titration kit• Harvest, titer and infect in a single day• Suitable for any AcMNPV-type baculovirusThe BacPAK qPCR Titration Kit provides an extremely fast and simple method for titrating your viral stocks. The kits use a quick DNA purification step before determining viral genome content using qPCR and SYBR® technologies (Figure 1). Whereas standard titration methods require up to 10 days to complete, this titration kit requires only 4 hours and works with any AcMNPV-based baculoviral vectors. Using qPCR dramatically shortens the time interval between viral harvest and target cell infection, allowing you to perform both on the same day. This means that you can avoid delays that lead to reduced viral infectivity and can infect target cells at a known multiplicity of infection (MOI) for more consistent results (1).
The BacPAK qPCR Titration Kit allows you to determine the viral genome copy number in baculoviral preparations from a calibrated DNA standard curve (Figure 2).
Figure 1. The BacPAK qPCR Titration Kit protocol.
Harvest baculoviralsupernatant
Purify viral DNA PerformqPCR Analyze data
100
Flu
ore
scen
ce (
dR
n)
40Cycle no.
10–1
10–2
0 5 10 15 20 25 30 35
24
Ct (
dR
n)
108
Initial quantity (copies)
6104 105 106 107
22
20
18
16
14
12
10
8
A
B
Figure 2. The BacPAK qPCR Titration Kit exhibits a wide dynamic range. The BacPAK DNA Control Template was serially diluted from 108 to 103 copies per sample and analyzed with the BacPAK qPCR Titration Kit. The amplification plots (Panel A) show a wide dynamic range of at least 6 orders of magnitude with no NTC (No-Template Control) background. The standard curve (Panel B) obtained by plotting the Ct values (determined from the amplification plots in Panel A) against the log of the DNA copy number in each sample, demonstrates a strong linear correlation between the Ct and the DNA copy number (log scale), with R2 = 1.000 and a PCR efficiency of 100%.
Comparison of BacPAK qPCR Titration to Other Titration Methods*
Titration Method Plaque Assay BacPAK Rapid Titer Assay BacPAK qPCR Titration
Description Count cleared plaques in infected cell monolayer
Immunostaining of Gp64 in infected cell monolayer
Measure viral DNA using SYBR qPCR with standard DNA as control
Time to Completion 1 week 48 hr 2–4 hr
Benefits Traditional, visual Simple, visual Fast, accurate
* Clontech offers two different kits for baculovirus titration: the BacPAK Baculovirus Rapid Titer Kit (Cat. No. 631406) utilizes a standard immunological assay to accurately identify virus-infected cells, and the BacPAK qPCR Titration Kit (Cat. No. 631414) measures viral DNA copies via SYBR qPCR.
The procedure is simple: viral DNA and BacPAK control DNA are serially diluted and subjected to qPCR. The DNA copy number of each viral sample is then determined by comparing its Ct value to a standard curve generated by plotting the Ct values of the diluted control samples against their respective copy numbers, as shown in Figure 2. With its simplicity, reproducibility, and short processing time, the BacPAK qPCR Titration Kit is ideal for determining baculoviral DNA titers.
References1. Rapid & Accurate Baculovirus Titration (2009) Clontechniques XXIV(3):8–9.
Product InformationBrand Product Size Cat.No
Clontech BacPAK Baculovirus Expression System each 631402
Clontech BacPAK6 DNA (Bsu36 I digest) 5 transfxns 631401
Clontech IPLB-Sf21 Insect Cells 1 vial 631411
Clontech BacPAK Grace’s Basic Medium 500 ml 631404
Clontech X-GLUC 100 mg 631721
Clontech In-Fusion Ready BacPAK Vector Set 3 vectors 631410
Clontech In-Fusion® HD Cloning System 50 rxns 639646
Clontech BacPAK Baculovirus Rapid Titer Kit 5 assays 631406
Clontech BacPAK qPCR Titration Kit 200 rxns 631414
10
I N C R E A S E D P R O T E I N Y I E L D A N D P U R I T Y
Features• Great for difficult proteins that can’t be expressed with the T7 system• Facilitates increased solubility due to expression at reduced temperature• Facilitates increased purity due to repressed expression of host proteins
Application• High-efficiency protein expression using a cold shock
promoter
DescriptionTakara’s pCold Expression Vectors offer Cold Shock expression technology for high purity, high yield protein production. The pCold series includes four different vectors.
M13 IG
pCold I4.4kb
pCold II4.4kb
pCold III4.4kb
pCold IV4.4kb
cspA 3'UTRmultiple cloning siteFactor Xa siteHis•TagTEEcspA 5'UTRlac operatorcspA promoter
ColE1 ori
Am
p la
cI
M13 IG
cspA 3'UTRmultiple cloning siteHis•TagTEEcspA 5'UTRlac operatorcspA promoter
ColE1 ori
Am
p
Am
p
Am
p
lacI
M13 IG
cspA 3'UTRmultiple cloning siteTEEcspA 5'UTRlac operatorcspA promoter
ColE1 ori
lacI
M13 IG
cspA 3'UTRmultiple cloning sitecspA 5'UTRlac operatorcspA promoter
ColE1 ori
lacI
In the following examples, genes that were poorly expressed or that produced insoluble protein with the T7 promoter expression system were expressed using the pCold system. pCold I DNA was used as an expression vector in E. coli. Expression from T7 promoter-driven vectors was induced with IPTG and T7 plasmid-containing cells were cultured at 37°C.
Expression of human gene A. Human gene A (~31 kDa) was expressed in both the T7 system and the cold-shock expression system. No expression was observed in the T7 system, but human gene A was expressed in the pCold system.
Expression of human gene C. Comparison of expression of soluble human gene C protein (~80 kDa) in the cold-shock expression sys-tem vs. the T7 system was performed. Target protein in the soluble fraction of pCold cells was dramatically higher than that of the T7 system.
N.C T7 pColdkDa
97.4
66.2
45
31
21.5
14.4
← Expression enabled
CBB staining of the entire protein fraction
T7 pCold T S T S
kDa
97.4
66.2
45
31
21.5
14.4
T: Entire protein fractionS: Soluble fraction
← Expression level increased
CBB staining
Takara’s pCold expression vectors
pCold Expression Vectors
Each includes the Cold Shock Protein A (cspA) promoter for expression of highly pure recombinant protein in E. coli at a high yield. These vectors selectively induce target protein synthesis at low temperature (15°C), a condition at which host protein synthesis is suppressed and protease activity is decreased. This results in high yields of 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). Three vectors also contain either a translation enhancing element (TEE), His-Tag sequence, and/or Factor Xa cleavage site. These vectors work equally well for synthesis of non-labeled and radiolabeled proteins and can be used with Takara’s Chaperone Plasmid Set (Cat. #3340).Reference1. Quing, G., et. al. (2004) Nature Biotechnol. 22(7):877-882.
Product InformationBrand Product Size Cat.No
Takara pCold Vector Set 1 Set (ea.5 µg) 3360
Takara pCold I DNA 25 µg 3361
Takara pCold II DNA 25 µg 3362
Takara pCold III DNA 25 µg 3363
Takara pCold IV DNA 25 µg 3364
11
pCold TF Vectors
Application
• Highly efficient protein expression using cold shock technology
• High yield of active protein due to use of Trigger Factor chaperone as a solubility-promoting fusion tag
Description
Takara’s pCold TF DNA Vector is a fusion cold shock expression vector that expresses Trigger Factor (TF) chaperone as a soluble fusion tag. Trigger Factor is a 45 kDa prokaryotic ribosome-associated chaperone protein that facilitates co-translational folding of nascent 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 multiple cloning 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 between TF-Taq and the Multiple Cloning Site (MCS).
pCold TF pColdpCold +
Chaperone T71 2 1 2 1 2 1 2
kDa
97
66
45
31
22
1. Cell extract solution
2. Soluble fraction
target protein
co-expressed
trigger factor**
Features
• Facilitates high yield protein expression with optimized protein folding• Enables expression of fusion proteins with a soluble tag to optimize solubility
Application
• Site-dependent cleavage of ssRNA
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 5’-ACA-3’ sequences.
pCold ProS2 DNA
mRNA Interferase™- MazF Enzyme
I N C R E A S E D P R O T E I N Y I E L D A N D P U R I T Y
Expression of protein A in T7 and pCold systems. The expression of enzyme protein A (~29 kDa) was poor when utilizing aT7 or pCold I expression systems, even when the pCold I construct was co-expressed with a chaperone. In contrast, the expression of target protein as a fusion (29 kDa + 52 kDa) was successful with pCold TF DNA, and most of the expressed protein was in soluble form. The expressed enzyme protein A showed activity even in the form of a fusion protein (data not shown).
These sequences facilitate tag removal from the expressed fusion protein. Most E. coli strains can serve as expression hosts.
DescriptionThe pCold Pro S2 expression vector features Protein S, a soluble tag from Myxococcus xanthus fused to the N-terminus of target proteins. Tight regulation of protein expression is maintained by a lac operator downstream of the cold shock promoter. HRV 3C Protease, Thrombin, and Factor Xa recognition sites are encoded between the Protein S tag and the MSC to facilitate tag removal.
This enzyme does not cleave double-stranded RNA, double-stranded DNA, or single-stranded DNA. mRNA Interferase-MazF is supplied as a fusion protein of E. coli MazF and Trigger Factor. The Trigger Factor protein is an E. coli chaperone protein. The enzyme is also supplied with a 5X MazF buffer (200 mM Sodium phosphate, pH 7.5, 0.05% Tween 20.)
Product InformationBrand Product Size Cat.No
Takara pCold TF DNA 25 µg 3365
Takara pCold™ ProS2 DNA 25 µg 3371
Takara mRNA Interferase™-MazF 1000 units 2415A
Application note
The pCold TF Protein Expression System Produces Soluble, Active Protein in E. coliwww.takara-bio.eu/appnote-pcold.pdf
12
Application
• Preferential expression of target protein by suppression of endogenous proteins using mRNA interferase plasmid
Description
This system utilizes an E. coli protein, MazF, described as an mRNA interferase by Suzuki et. al. MazF is a sequence-specific endoribonuclease that cleaves single strand RNAs at 5’-ACA-3’ (ACA) sequences. When using the SPP System, the transcript of interest should therefore lack ACA sequences. MazF is co-expressed in the E. coli host and suppresses expression of non-target genes by cleaving host transcripts at ACA sequences. Therefore, the target protein is the most abundantly expressed protein (Figure 1). Because of the requirement for target gene transcripts to lack ACA sequences, the SPP System is not suitable for all genes of interest; however, when appropriate, it can result in extremely high levels of protein production.
ReferenceSuzuki, M., et. al. (2005) Molecular Cell 18(2)253-261.
Figure 1. Synthesis of cspA-promoter expressed envAB in presence and absence of MazF. E. coli BL21 cells co-expressing MazF and pCold (SP-4) envZB showed good envZB expression and extremely low background synthesis of host proteins.
SPP System
I N C R E A S E D P R O T E I N Y I E L D A N D P U R I T Y
Product InformationBrand Product Size Cat.No
Takara SPP SystemTM I 1 kit 3367
Takara SPP SystemTM II 1 kit 3368
Takara SPP SystemTM III 1 kit 3369
Takara SPP SystemTM IV 1 kit 3370
Takara SPP SystemTM I-IV 1 kit 3366
13
C E L L F R E E P R O T E I N E X P R E S S I O N
Features
• Easy-to-use system allows generation of protein in as little as 1 h in a single-tube reaction• Provides higher yield of functional protein and greater consistency than rabbit reticulocyte or wheat germ in vitro translation systems• Excellent with challenging proteins such as large proteins (over 150 kDa) and proteins requiring post-translational modification • Amenable to high-throughput screening; bulk sizes available
Applications
• Expression of toxic proteins that are lethal to host cells of in vivo expression systems
• Rapid analysis of protein function
• Rapid analysis of mutation series or truncation series: generate protein quickly and assess functionality using your downstream assay
• High-throughput proteomic studies
• Expression of proteins that are easily degraded or insoluble in conventional in vivo expression systems such as E. coli
Description
The Human Cell-Free Protein Expression System from Takara Bio is easy to use. The single-tube reaction is easily assembled and protein synthesis is complete in as little as 1 h at 32°C. The Human Cell-Free Protein Expression System provides high yields (e.g., 50 µg/ml of human eIF4G) of functional protein, including proteins requiring modifications such as glycosylation, phosphorylation, or fatty acylation. Excellent yield is observed even with large proteins (over 150 kDa). Genes of interest may be cloned rapidly into pT7-IRES vectors using In-Fusion cloning technology. After expression, proteins with N-terminal or C-terminal His-tag or N-terminal Myc tag can be generated, depending on the choice of vector. Bulk sizes are available for high-throughput studies; contact custom@takara-bio.eu for more information.
In vitro translation has many advantages for protein expression: it is excellent for rapid studies of protein function or features, amenable to high-throughput studies, useful for proteins that are degraded or insoluble with in vivo systems, and can be used to generate lethal proteins that cannot be expressed using in vivo systems due to toxicity. In contrast to expression using prokaryotic host cells,
Human Cell-Free Expression System
EMCVIRES
Ampr
ColE1 ori
His-TagFactor Xa Site
Multiple Cloning SitepolyA T7 terminator
pT7-IRES His-N DNA(3,429 bp)
in vitro translation systems also allow post-translational modifications such as glycosylation, phosphorylation, and fatty acylation.
pT7-IRES Vector Information
Vectors in the pT7-IRES series include a T7 promoter and EMCV IRES sequence to facilitate transcription and translation in the Human Cell-Free Protein Expression System, as well as a Multiple Cloning Site (MCS), convenient tags (N- or C-terminal His-tag or Myc tag sequences), a Factor Xa protease cleavage site for tag removal, poly-A signal, and T7 terminator.
• pT7-IRES His-N DNA (Cat. # 3290) encodes an N-terminal His-tag
• pT7-IRES His-C DNA (Cat. # 3291) encodes a C-terminal His-tag
• pT7-IRES Myc-N DNA (Cat. # 3292) encodes an N-terminal c-Myc tag
Related In-Fusion Cloning Products
For rapid and easy cloning, use the In-Fusion HD Cloning System to generate pT7-IRES constructs with your insert of interest. See page 15.
Map of pT7-IRES vector.
14
0 1 2 3 4 5 6 7 8
120
100
80
60
40
20
0
Relative absorbance
Time (hour)
Mixture-2(ー)Mixture-2(+)
Time course of β-galactosidase expression and effect of translation enhancement factor. Eight replicates of β-galactosidase in vitro translation were performed using 1 µL of Control Vector per reaction. At each of the indicated time points (0, 0.5, 1, 2, 3, 4.5, 6, and 8 h after start of the reaction), one reaction tube was removed and used for β-galactosidase activity assay with O-nitrophenyl-β-D-galactopyranoside (ONPG) as the substrate. A separate set of reactions were conducted in absence of the translation enhancement factor (Mixture-2). The activity of β-galactosidase increased over time to peak at approximately 4.5 h. Additionally, the presence of Mixture-2 containing translation enhancement factor markedly increased yield of active protein.
Principle of the Human Cell-Free Protein Expression System. A) In an easy and simple protocol, target protein translation is initiated by adding pT7-IRES Vector containing the target gene cassette and other kit components. B) The target gene RNA transcribed from the pT7-IRES Vector has an IRES sequence designed to promote protein translation initiation. As protein synthesis progresses, the translation initiation factor from the cell lysate becomes inactivated. The transla-tion enhancement factor in the reaction mixture, however, reactivates this inactivated translation initiation factor and thereby maintains a high level of translation.
1 2 3
◄ ◄
C E L L F R E E P R O T E I N E X P R E S S I O N
Synthesis of high molecular weight proteins using the Human Cell-Free Protein Expression System. In vitro translation reactions were performed to synthesize human Dicer (200 kDa, lane 2) or human eIF4G (170 kDa, lane 3) protein. Reactions were analyzed by SDS-PAGE and Coomassie blue staining. Arrowheads indicate target proteins. Lane 1, negative control.
Product InformationBrand Product Size Cat.No
Takara Human Cell-Free Protein Expression System 10 rxns 3281
Takara pT7-IRES His-N DNA 20 µg 3290
Takara pT7-IRES His-C DNA 20 µg 3291
Takara pT7-IRES Myc-N DNA 20 µg 3292
(continued...)
15
Optimal PCR-cloning method for protein expression
A simple 15-30 minute In-Fusion® reaction results in the creation of seamless and precisely engineered constructs, where no extra bp of vector or restriction-site-derived DNA is added. This is a crucial advantage when generating constructs to express tagged proteins as no undesirable vector or restriction-site-derived amino acids are added to the expressed protein, unless specifically required by you.
This allows the expressed tagged protein to mimic the native protein as closely as possible, in order to aid its correct folding, especially important in studies of protein function.
Amplify yourgene of interest
Gene-specific primerswith 15 bp extensions
homologous to vector ends
The In-Fusion enzymeproduces single-stranded
PCR fragment and vector endsthat are fused due to the
15 bp homology
PCRproduct
Anylinearized
vector
Single-tube protocol
x x
Recombinant vector
In-Fusion allows seamless, directional cloning of PCR products directly into your vector of choice and generates more than 90% correct clones.
Product name Cells included?Cloning Enhancer
included?Nucleospin columns
included?Size (rxns) Cat. No.
Liquid format
In-Fusion HD Cloning Kit 10; 50; 100639648; 639649; 639650
In-Fusion HD Cloning System CE
Yes Yes 10; 50; 100639636; 639637; 639638
In-Fusion HD Cloning System Yes Yes 10; 50; 100639645; 639646; 639647
Dry-down format
In-Fusion HD EcoDry™ Cloning Kit
8; 24; 96639689; 639690; 639691
In-Fusion HD EcoDry™ Cloning System
Yes Yes 8; 24; 48; 96639684; 639685; 639686; 639688
For complete list of In-Fusion products available please see www.takarabio.com
PCR enzymes perfect for In-Fusion® cloning!In-Fusion cloning can be performed with inserts generated by any PCR enzyme independent of whether they are sticky or blunt ended PCR products. Clontech’s Advantage HD is a high fidelity enzyme suitable also for use with small amounts of template DNA. Takara’s PrimeSTAR® enzymes are also recommended for use with In-Fusion due to their capacity for combining high fidelity with long and GC-rich targets or speed.
PrimeSTAR GXL is the best commercially available enzyme for amplifying long targets (up to 30 kb) and can be used with excess as well as GC/AT-rich template DNA .Takara’s high fidelity and high speed enzyme, PrimeSTAR® Max, has the fastest extension speed available (≤ 5 sec./kb), along with extremely high accuracy, specificity and sensitivity.
Product InformationBrand Product Size Cat.No
Clontech Advantage HD 200 rxns 639241
Takara PrimeSTAR GXL DNA Polymerase 250 Units R050A
1000 Units R050B
Takara PrimeSTAR Max DNA Polymerase 100 rxns R045A
400 rxns R045B
C L O N I N G M E T H O D F O R P R O T E I N E X P R E S S I O N
16
Brand Vector Expression (1,2) Tag/marker (3) Cleavage Size
(kb) Promoter Selection Prok.
Selection Euk. MCS Replication origin Sold as
part ofSingle cat no
E. Coli Expression vectors
Takara pCold™ I (SP-4) DNA His Factor Xa 4.4 cspA AmpR Yes ColE1 (high copy)3360 3366 3367
3361
Takara pCold™ II (SP-4) DNA His 4.4 cspA AmpR Yes ColE1 (high copy)3360 3366 3368
3362
Takara pCold™ III (SP-4) DNA 4.4 cspA AmpR Yes ColE1 (high copy)3360 3366 3369
3363
Takara pCold™ IV (SP-4) DNA 4.4 cspA AmpR Yes ColE1 (high copy)3360 3366 3370
3364
Takara pCold™ TF DNA HisHRV 3C, Thrombin, Factor Xa
5.7 cspA AmpR Yes ColE1 (high copy) 3365
Takara pCold™ ProS2 DNA HisHRV 3C, Thrombin, Factor Xa
5 cspA AmpR Yes ColE1 (high copy) 3371
Takara pMazF DNA 4.8 lac CmR No pACYC (low copy)
3366 3367 3368 3369 3370
Not sold separately
Clontech pET6xHN-N C 6xHN Enterokinase 5.8 T7 lac AmpR N/A Yes pBR322 (low copy) 631431 631430
Not sold separately
Clontech pET6xHN-C C 6xHN Thrombin 5.8 T7 lac AmpR N/A Yes pBR322 (low copy) 631431 631430
Not sold separately
Clontech pET6xHN-N C 6xHN Enterokinase 5.8 T7 lac AmpR N/A In-Fusion Ready pBR322 (low copy) 631429
631428Not sold separately
Clontech pET6xHN-C C 6xHN Thrombin 5.8 T7 lac AmpR N/A In-Fusion Ready pBR322 (low copy) 631429
631428Not sold separately
B. Subtilis Expression vectors
Takara pBE-S DNA 5.9 aprE AmpR KanR Yes ColE1+pUBori 3380 Not sold separately
Baculovirus Expression vectors
ClontechIn-Fusion Ready pBacPAK-Cterm 6xHN
C 6xHN Thrombin 5.4 Polyhedrin AmpR N/A In-Fusion Ready pUC (high copy) 631410
ClontechIn-Fusion Ready pBacPAK-Nterm 6xHN
C 6xHN Enterokinase 5.4 Polyhedrin AmpR N/A In-Fusion Ready pUC (high copy) 631410
Clontech pBacPAK8 C None None 5.5 Polyhedrin AmpR N/A Yes pUC (high copy) 631402
Clontech pBacPAK9 C None None 5.5 Polyhedrin AmpR N/A Yes pUC (high copy) 631402
Brevibacillus Vectors
TakarapNC-HisE DNA, Secretory
His Enterokinase 5,2 P2 AmpR NeoR Yes ColE1+Brevibacillus HB123
Takara pNC-HisF DNA, Secretory His Factor Xa 5,2 P2 AmpR NeoR Yes ColE1+Brevibacillus HB122
Takara pNC-HisT DNA, Secretory His Thrombin 5,2 P2 AmpR NeoR Yes ColE1+Brevibacillus HB121
Takara pNCMO2 DNA, Secretory
5,2 P2 AmpR NeoR Yes ColE1+Brevibacillus HB200 HB112
Takara pNI DNA, Intracellular 5 P2 AmpR NeoR Yes ColE1+Brevibacillus HB131
Takara pNI-His DNA, Intracellular His Enterokinase 5,1 P2 AmpR NeoR Yes ColE1+Brevibacillus HB132
Takara pNY326 DNA, Secretory
3,4 P5 NeoR Yes Brevibacillus HB200 HB111
Mammalian Expression vectors
TakarapBApo-CMV Neo DNA (also for miRNA, adeno)
C None 4.6 CMV IE AmpR NeoR Yes High copy 3240
TakarapBApo-CMV Pur DNA (also for miRNA, adeno)
C None 4.4 CMV IE AmpR PuroR Yes High copy 3241
E X P R E S S I O N V E C T O R S O V E R V I E W
1) FP: Fluorescent Protein 2 ) C: Constitutive 3) I: Inducible
17
E X P R E S S I O N V E C T O R S O V E R V I E W
Brand Vector Expression (1,2) Tag/marker (3) Cleavage Size
(kb) Promoter Selection Prok.
Selection Euk. MCS Replication origin Sold as
part ofSingle cat no
TakarapBApo-CMV DNA (also for miRNA, adeno)
C None 3.4 CMV IE AmpR None Yes High copy 3242
TakarapBApo-EF1-α Neo DNA (also for miRNA, adeno)
C None 5.2 EF-1α AmpR NeoR Yes High copy 3243
TakarapBApo-EF1-α Pur DNA (also for miRNA, adeno)
C None 5 EF-1α AmpR PuroR Yes High copy 3244
Takara pT7-IRES His-N DNA His Factor Xa 3.4 T7 AmpR Yes 3290
Takara pT7-IRES His-C DNA His Factor Xa 3.4 T7 AmpR Yes 3291
Takara pT7-IRES Myc-N DNA Myc Factor Xa 3.4 T7 AmpR Yes 3292
Clontech pCMV-HA-C C HA 3.8 CMV IE AmpR None Yes pUC (high copy) 635690
Clontech pCMV-HA-N C HA 3.8 CMV IE AmpR None Yes pUC (high copy) 631604 635690
Clontech pCMV-Myc-C C myc 3.8 CMV IE AmpR None Yes pUC (high copy) 635689
Clontech pCMV-Myc-N C myc 3.8 CMV IE AmpR None Yes pUC (high copy) 635689 631604
Clontech pEF1-α-Myc C myc None 4.6 EF-1α AmpR None Yes pUC (high copy) 631991
Clontech pEF1-α-HA C HA None 4.6 EF-1α AmpR None Yes pUC (high copy) 631992
Clontech pCMV-(DYKDDDDK)-C C DYKDDDDK
(FLAG) 3.8 CMV AmpR None Yes pUC (high copy) 635688
Clontech pCMV-(DYKDDDDK)-N C DYKDDDDK
(FLAG) 3.8 CMV AmpR None Yes pUC (high copy) 635688
Clontech Fluorescent Protein Vectors - Fusion C FP 4.7 CMV IE KanR NeoR Yes pUC (high copy) Multiple
Clontech C FP 5.5 EF-1α KanR NeoR Yes pUC (high copy) Multiple
ClontechFluorescent Protein Vectors - Fusion, lentiviral
C FP 8,8; 9,5 CMV IE AmpR PuroR Yes pUC (high copy) Multiple
ClontechFluorescent Protein Vectors - Fusion, lentiviral
C FP 9.5 EF-1α AmpR PuroR Yes pUC (high copy) Multiple
Clontech Fluorescent Protein Vectors - bicistronic C FP 5.3 CMV IE KanR NeoR Yes pUC (high copy) Multiple
Clontech C FP 6 EF-1α KanR NeoR Yes pUC (high copy) Multiple
ClontechFluorescent Protein Vectors - bicistronic, lentiviral
C FP 8,2; 8,9 CMV IE AmpR None Yes pUC (high copy) Multiple
ClontechFluorescent Protein Vectors - bicistronic, lentiviral
C FP 8.9 EF-1α AmpR None Yes pUC (high copy) Multiple
Inducible Mammalian Expression Systems
Clontech Tet-ON 3G I None None 3.4 TRE3G AmpR None Yes pUC (high copy) 631168 Not sold separately
Clontech Tet-OFF Advanced I None None 2.6 TRE-Tight AmpR None Yes Col E1 (low copy) 631059
Clontech Tet-On 3G - EF1-α Version I None None 3.4 TRE3G AmpR None Yes pUC (high copy) 631167 Not sold
separately
Clontech Tet-On 3G - Bidirectional I With or
without FP2,9; 3,6 TRE3G-BI AmpR None Yes pUC (high copy) multiple Not sold
separately
Clontech Tet-On 3G - Bicistronic I With or
without FP 4; 4,7 TRE3G AmpR None Yes pUC (high copy) multiple Not sold separately
Clontech Tet-On 3G - Lentiviral: pLVX-TRE3G I None None 7.8 TRE3GV AmpR None Yes pUC (high copy) 631187 Not sold
separately
ClontechTet-On 3G - Retroviral: pRetroX-TRE3G
I None None 6.6 TREGV AmpR PuroR Yes pUC (high copy) 631188 Not sold separately
ClontechTet-On 3G - Adenoviral: pAdenoX-Tet3G
I None None 36 TRE3G AmpR None In-Fusion Ready pUC (high copy) 631180 Not sold
separately
Clontech Tet-Express System I With or without FP None
TRE3G, TRE3G-BI, TRE3GV
AmpR None Yes pUC (high copy) multiple Not sold separately
1) FP: Fluorescent Protein 2 ) C: Constitutive 3) I: Inducible
18
F O L D I N G
Application• Promotes correct in vivo folding of expressed recombinant
proteins in E. coli
DescriptionThe Chaperone Plasmid Set consists of 5 different plasmids, each of which is designed to express multiple molecular chaperones. Together, they function as a “chaperone team” to facilitate protein folding. Co-expression of a target protein with one of these plasmids 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 with an ampicillin resistance gene as a marker. The chaperone genes are situated downstream of an araB or Pzt-1 (tet) promoter. Therefore, 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.
Note that this system cannot be used in combination with chloramphenicol-resistant E. coli host strains or expression plasmids that carry a chloramphenicol-resistance gene. For example, E. coli BL21(DE3), which is often used with pET systems, is a compatible host strain. However, E. coli BL21(DE3) pLysS and BL21(DE3) pLysE, which contain pLysS or pLysE plasmids that have the pACYC replication origin and the Cmr gene, cannot be used with this system.
pKJE77.2 kb
Cmr
araC pACYC ori
araB
dnaK dnaJ
grpE
pACYC ori
groEL
pGro75.4 kb
Cmr
araC
araB
groES
rrnBT1T2
pACYC ori
Pzt1pG-KJE8
11.1 kb
CmraraC
tetR
araB
dnaK
dnaJgrpE
groES
groEL
pACYC ori
tig
pTf165 kb
Cmr
araC
araBpACYC ori
pG-Tf28.3 kb
Cmr
tetRtig
groES
groEL
Pzt1
The combination of Cold Shock Expression Vectors and the Chaperone Plasmid Set often leads to significant improvement in expression lev-el of soluble forms of target proteins. If sufficient expression or solubilization cannot be achieved using pCold vectors alone, we recommend co-expression with chaperone plasmids. Furthermore, pCold vector-based expression systems may produce better results by co-expressing chaperone plasmids carrying the tig sequence, such as pG-Tf2 or pTf16, which are included in the Chaperone Plasmid Set (data not shown).
Chaperone Plasmid Set
Plasmids with the Chaperone Plasmid Set work well in combination with the pCold expression system vectors.
Resistance
Plasmid Chaperone Promoter Inducer Marker
pG-KJE8 dnaK-dnaJ-grpE-groES-groEL araB, Pzt1 L-Arabinose, Tetracycline Cmr
pGro7 groES-groEL araB L-Arabinose Cmr
pKJE7 dnaK-dnaJ-grpE araB L-Arabinose Cmr
pG-Tf2 groES-groEL-tig Pzt1 Tetracycline Cmr
pTf16 tig araB L-Arabinose Cmr
Human gene A (~70 kDa) was expressed in insoluble form when using pCold I alone. However, the level of soluble expressed protein increased significantly when the chaperone plasmid pG-Tf2 was co-expressed with the pCold I construct.
Human gene B (~24 kDa) was not expressed when using pCold I DNA alone. However, co-expression with the chaperone plasmid pG-Tf2 resulted in expression of high levels of target protein in soluble form.
Product InformationBrand Product Size Cat.No
Takara Chaperone Plasmid Set 1 Set 3340
References1. Nishihara, K., et al. (2000) Microbiol. 66(3):884-889.2. Nishihara, K., et al. (1998) Appl. Environ. Microbiol. 64(5):1694-1699.
Related products
Chaperone Competent Cells: Chaperone Competent Cell pG-KJE8/BL21 (Cat. #9121), Chaperone Competent Cell pG-Tf2/BL21 (Cat. #9124), Chaperone Competent Cell pGro7/BL21 (Cat. #9122), Chaperone Competent Cell pKJE7/BL21 (Cat. #9123), Chaperone Competent Cell pTf16/BL21 (Cat. #9125), Chaperone Competent Cells BL21 Set (Cat. #9120).
19
F O L D I N G
Guanidine hydrochloride
unfolds inclusion bodies
Surfactants prevent protein
aggregation
Highly polymerized CA removes surfactants
and facil i tates protein refolding
Biologically active protein in
thermodynamically stable native conformation
Principle of the Refolding CA Kit
Chaperonin GroE
Application• Facilitates refolding of denatured proteins
DescriptionChaperonin GroE is a protein complex composed of GroEL (14 subunits, 57 kDa) and GroES (7 subunits, 10 kDa).
Corystein™ (Purothionin) ReagentApplication
• Facilitates protein refolding by promoting exchange reactions between disulfide bonds
DescriptionCorystein™ (Purothionin) Reagent is a polypeptide purified from wheat endosperm. It catalyzes the formation of correct disulfide bonds in pro teins. Corystein™ Reagent can be used alone or together with thioredoxin on a variety of proteins to re-form disulfide bonds.
Refolding CA KitApplication
• 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 allows identification of the best conditions for correct protein folding and restoration of protein activity.
The Small Kit (Cat. #7350) is supplied with guanidine hydrochloride and DTT for protein denaturation, four different surfactants that can be added independently to the unfolded protein solution to protect 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. The resulting supernatant contains the refolded protein.
The Large Kit (Cat. #7351) is used for large-scale refolding after initial determination of with the Small Refolding CA kit, and consists only of denaturant and CA.
References1. Machida, S., et al. (2000) FEBS Lett. 486(2):131-135.2. Sundari, C.S., et al. (1999) FEBS Lett. 443(2):215-219.3. Daugherty, D.L., et al. (1988) J. Biol. Chem 273(51):33961-33971.
It is thought to support the ability of proteins to form tertiary structure upon or immediately after translation. GroE is essential to assembly (and presumably reassembly after denaturation) of protein complexes in vivo. Chaperonin GroEL and GroES can be used for refolding denatured proteins to recover functional activity.
Product InformationBrand Product Size Cat.No
Takara Chaperonin Gro EL 5 mg 7330
Takara Chaperonin Gro ES 0.5 mg 7331
Takara Corystein™ (Purothionin) Reagent 5 mg 7311
Takara Refolding CA Kit 25 reactions 7350 (small)
Takara Refolding CA Kit 1 kit 7351 (large)
Application note
Unfolding the Potential of ProteinsNew refolding technologies are essential for tomorrow’s recombinant proteins, By Joby Marie Chesnickwww.takara-bio.eu/appnote-protfolding.pdf
20
H I S T A G G E D P R O T E I N P U R I F I C A T I O N
Talon® Co Resin-Highest Purity
Advantages
• Highest purity• Highest specificity for his-tagged proteins• Reduced co-purification of impuritiesEach TALON reactive core is charged with a cobalt ion that has a much higher selectivity for histidine tags than nickel ions. Only proteins containing adjacent histidines or specially positioned histidines are able to bind to TALON. In contrast, the spatial requirement for nickel-based resins is less strict, so using Ni-NTA resins often results in co-purification of contaminants (see figure).
TALON resin is designed to maximize your yield of biologically active protein. Choose TALON when purity is of utmost importance or when purifying under native conditions.
Applications• Crystallography• Functional assays• Structural & functional investigations
IMAC purification—TALON vs. Ni-NTA. 6xHN-AcGFP1 was purified from Sf21 cells using TALON or Ni-NTA. The chromatogram for each column is shown. The absorbance at 475 nm indicates the amount of target protein (AcGFP1) present in each fraction.
A47
5 (A
U)
0Time (min)
0.01
Load
0.04
0.03
0.02
10 20
Load
Was
h
Co
nta
min
ant
pea
kis
sep
arat
ed
Was
h
Elu
te
Elu
te
A47
5 (A
U)
0Time (min)
TALON Ni-NTA
10 20 30 40
0.01
0.04
0.03
0.02
Co
nta
min
ant
pea
kco
elu
tes
His60 Ni Superflow™ Resin-Highest Capacity
Advantages
• Highest binding capacity (60 mg/ml)• Purify proteins under native or denaturing conditions• Low leakage of Ni2+ ionsHis60 Ni Superflow Resin is a high-capacity resin for the efficient purification of his-tagged proteins. The resin enables fast, easy, and reproducible chromatographic separations and can be re-used for the same protein. His60 Ni Resin is compatible with batch/gravity-flow applications as well as with the major automated liquid chromatography systems and manual syringe processing.
In one application, His60 Ni Superflow Resin performance was compared to the performance of Competitor Q’s Ni Superflow resin. 6xHN-AcGFP1 was purified from equivalent amounts of the same sample, following each manufacturer’s protocol. Clontech’s pEcoli Linear Expression System was used to express 6xHN-AcGFP1 in E. coli. Higher yields & better purity were obtained using our His60 Ni Superflow Resin (see figure).
Applications• Protein labeling• Immunization to produce antibodies• Animal studies
A
B
His60 Ni Superflow Outperforms Ni-NTA. Superior yields and better purity were obtained when comparing His60 Ni Superflow (Panel A) to Competitor Q’s Ni resin (Panel B).
His60 Ni Resin M OS FT W1 W2 E1 E2 E3 E4
NI-NTA Superflow Resin M OS FT W1 W2 E1 E2 E3 E4
kD
150 – 100 – 75 –
50 – 37 –
25 – 20 – 15 –
kD
150 – 100 – 75 –
50 – 37 –
25 – 20 – 15 –
6xHN-AcGFP1
6xHN-AcGFP1
Higher purity
Lower purity
Higher yield
Lower yield
21
H I S T A G G E D P R O T E I N P U R I F I C A T I O N
TALON Resin and Magnetic Beads
TALON Resin• Use for batch/gravity applications• Highest purity of your target protein
TALON Superflow Resin• Use for FPLC applications• Highest purity of your target protein
TALON CellThru Resin• Purify proteins directly from crude cell lysates, sonicates, and fermentation
liquids• Ideal for purification of membrane-bound proteins and multiprotein complexes
TALON Magnetic Beads• Microscale purification or screening of his-tagged proteins (bead particle size:
20–75 µm)• Elute in small volumes (50–200 µl)
His60 Ni Superflow Resin• Use for batch/gravity and FPLC applications• Highest binding capacity—60 mg/ml• Superior performance compared to other Ni-based resins
(highest capacity and better purity)• Low nickel ion leakage• Purify under native or denaturing conditions• Many sizes are available, from 10 ml to 1,000 ml
Buffers• HisTALON Buffer Set: complete buffer set for extraction and purification
of his-tagged proteins using TALON resin• His60 Ni Buffer Set: complete buffer set for extraction and purification
of his-tagged proteins using His60 Ni Superflow resin• xTractor Buffer: optimized lysis buffer for recombinant protein extraction• xTractor Buffer Kit: complete extraction kit with lysozyme and DNase I,
for efficient extraction of high molecular weight proteins
Detection of His-Tags
Antibodies• 6xHis mAb-HRP conjugate• 6xHis Monoclonal Antibody (Albumin Free)• 6xHN Polyclonal Antibody
His60 Ni Resin
Buffers and Detection Reagents
22
1 ml & 5 ml Cartridges FPLC Cartridges—HisTALON™ & His60 NiReady-to-use 1 ml and 5 ml cartridges
• Use on any chromatography system• Simple manual operation using a syringe• Can be connected in series to scale-up
HisTALON Superflow His60 Ni Superflow
Gravity Columns Gravity Columns—HisTALON & His60 NiReady-to-use 1 ml columns
• Up to 60 mg pure his-tagged protein per His60 Ni Superflow column
• Up to 20 mg highly pure his-tagged protein per HisTALON column
HisTALON His60 Ni Superflow
TALON Single Step Columns Single Step Columns—TALON Single StepReady-to-use 5 ml and 20 ml columns
• Load bacterial culture directly onto column• On-column extraction and purification• Load bacterial culture, wash, and elute target
protein
5 ml 20 ml
TALONSpin Columns Disposable Spin Columns—TALONSpin™Ready-to-use prepacked spin columns for small-scale purification
• Rapid, parallel purification in as little as 30 minutes
• Applications: screening of positive clones, trial-level purification
TALON 2 ml Disposable Gravity Columns Empty 2 ml Disposable Columns—TALONReady-to-use empty 2 ml gravity columns
• Fill columns with TALON, TALON Superflow, or His60 Ni Superflow resins
• Also suitable for glycoprotein enrichment, phosphoprotein & phosphopeptide enrich-ment, GST-tag, and antibody purification resins
CellThru 10 ml Disposable Columns Empty 10 ml Disposable Columns—CellThruReady-to-use empty 10 ml gravity columns
• Fill columns with TALON CellThru, TALON, TALON Superflow, or His60 Ni Superflow resins
• Also suitable for glycoprotein enrichment, phosphoprotein & phosphopeptide enrich-ment, and GST-tag & antibody purification resins
H I S T A G G E D P R O T E I N P U R I F I C A T I O N
23
Clontech Protein Purification Products InformationProduct Resin type Size Cat.No
TALON Resin
TALON Metal Affinity Resin Cobalt
10 ml25 ml100 ml250 ml2 x 250 ml4 x 250 ml
635501635502635503635504635652635653
TALON Superflow Metal Affinity Resin Cobalt
25 ml100 ml250 ml2 x 250 ml4 x 250 ml
635506635507635670635669635668
TALON CellThru Cobalt10 ml100 ml
635509635510
TALON Magnetic Beads Cobalt2 x 1 ml6 x 1 ml
635636635637
TALON Prepacked
HisTALON Gravity Columns Cobalt 5 columns 635655
HisTALON Gravity Columns Purification Kit Cobalt 20 purifications 635654
TALONspin Columns Cobalt10 columns25 columns50 columns
635601635602635603
TALON Single Step Columns (5 ml) Cobalt2 columns25 columns
635631635628
TALON Single Step Columns (20 ml) Cobalt 10 columns 635632
HisTALON Superflow Cartridges (5 x 1 ml) Cobalt 5 cartridges 635650
HisTALON Superflow Cartridge (1 x 5 ml) Cobalt 1 cartridge 635683
HisTALON Superflow Cartridges (5 x 5 ml) Cobalt 5 cartridges 635682
HisTALON Superflow Cartridge Purification Kit (5 x 1 ml) Cobalt 20 purifications 635649
HisTALON Superflow Cartridge Purification Kit (5 x 5 ml) Cobalt 5 purifications 635681
TALON Disposable Columns
CellThru 10-ml Disposable Columns - 20 columns 635513
TALON 2 ml Disposable Gravity Column - 50 columns 635606
His60 Ni Superflow Resin
His60 Ni Superflow Nickel 10 ml25 ml4 x 25 ml250 ml2 x 250 ml4 x 250 ml
635659635660635661635662635663635664
His60 Ni Prepacked
His60 Ni Gravity Columns Nickel 5 columns 635657
His60 Ni Gravity Columns Purification Kit Nickel 20 purifications 635658
His60 Ni Superflow Cartridges (5 x 1 ml) Nickel 5 cartridges 635675
His60 Ni Superflow Cartridge (1 x 5 ml) Nickel 1 cartridge 635680
His60 Ni Superflow Cartridges (5 x 5 ml) Nickel 5 cartridges 635679
His60 Ni Superflow Cartridge Purification Kit (5 x 1 ml) Nickel 20 purifications 635674
His60 Ni Superflow Cartridge Purification Kit (5 x 5 ml) Nickel 5 purifications 635678
H I S T A G G E D P R O T E I N P U R I F I C A T I O N
24
O T H E R P R O T E I N P U R I F I C A T I O N
The FLAG tag is commonly known as the DYKDDDDK epitope tag and is used as an N- or C- terminal epitope tag in many fusion proteins because it is small and therefore unlikely to alter the biochemical properties or localization of the protein to which it is fused.The anti-DYKDDDDK Antibody can be used for the detection of FLAG-tagged proteins. This monoclonal antibody recognizes the well-known FLAG epitope tag and detects N- or C-terminally tagged FLAG fusion proteins on Western blots. The highly specific and sensitive antibody does not cross-react with endogenous proteins.Anti-DYKDDDDK Beads consist of a monoclonal anti-DYKDDDDK antibody, crosslinked to agarose beads. These beads are used to purify or immunoprecipitate DYKDDDDK-tagged or FLAG-tagged fusion proteins from cell lysates. Clontech’s Anti-DYKDDDDK Beads and Immunoprecipitation
DYKDDDDK (FLAG Epitope) Immunoprecipitation and Detection
Anti-DYKDDDDK Beads for immunoprecipitation provide higher purity and lower background when compared with competitor S anti-FLAG beads.
Figure 1. GST-tagged protein purification from whole cell extract. Whole cell extracts containing GST-DHFR (Panel A) and GST alone (Panel B) were loaded, washed, and eluted from glutathione resin columns. The resulting purification fractions were analyzed by SDS-PAGE (upper panels) and Western blot (lower panels) with an anti-GST IgG. WCE = whole cell extract. FT = flowthrough. ELU = eluate.
5 15 25
1.0
0.0
Effluent (ml)
Ab
sorb
ance
(25
4 n
m)
I II
Elu
te
Load
Wash
FT ELU
WC
E
GST-DHFR
Western
kDa
205
55
20
6.5
A
4 8 10 20 30
1.0
0.0
Effluent (ml)
Ab
sorb
ance
(25
4 n
m)
I II
Elu
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Wash
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WC
E
GST
Western
BkDa
205
55
20
6.5
GST-Tag Purification Resins• One-step isolation of highly pure GST-tagged proteins• High binding capacity (>10 mg tagged protein per ml resin)• Available in flexible formats for gravity flow & FPLC
applications• Easily regenerated for reuse & competitively priced
Flexible Resin FormatsGlutathione-Superflow and -Uniflow Resins bind GST (glutathione-S-transferase) tags with high affinity and specificity, allowing rapid, efficient purification of GST-tagged proteins. These resins are based on 6% and 4% cross-linked agarose,
respectively, with glutathione covalently bound to the resins.Glutathione-Superflow Resin is suitable for FPLC applications. It can withstand higher flow rates and back pressure with flow rates as high as 15 ml/cm2/min. Alternatively, Glutathione-Uniflow Resin, with a maximum linear flow rate of 2 cm2/min, is suitable for purification of large fusion proteins using batch/gravity-flow purification or standard chromatography methods.For greater convenience, the GST Purification Kit provides sufficient stock buffers and prepacked Glutathione-Uniflow Columns for performing 5 batch/gravity-flow purifications. Up to 10 mg of GST-tagged protein per column can be purified using this kit.
Buffer Set can be purchased separately to make a complete Co-IP kit for purification of FLAG-tagged proteins.
Product InformationBrand Product Size Cat.No
Clontech Glutathione-Superflow Resin 10 ml 635607
Clontech Glutathione-Superflow Resin 100 ml 635608
Clontech GST Purification Kit 5 purifications 635619
Product InformationBrand Product Size Cat.No
Clontech Anti-DYKDDDDK Antibody 200 µg 635691
Clontech Anti-DYKDDDDK Beads 1 ml 635686
Clontech Immunoprecipitation Buffer Set 30 rxns 635687
Clontech pCMV-DYKDDDDK Vector Set 10 µg each 635688
25
c-Myc Monoclonal Antibody The c-myc monoclonal mouse antibody recognizes an epitope located within residues 410–419 of the human p62-c-Myc protein.This antibody is suitable for immunoprecipitation of c-myc-tagged fusion proteins from cell lysates, ELISA, immunocytochemistry of microinjected or transfected cells, and localization of c-myc-tagged proteins.
c-Myc and HA-tagged proteins Detection and purification
HA-Tag Polyclonal AntibodyThe HA-tag antibody was raised in rabbits against a synthetic HA peptide (YPYDVPDYA, the hemagglutinin epitope of human influenza A virus) and purified using protein A. This purified antibody is suitable for Western blotting, immunoprecipitation, immunofluorescence, and ELISA applications.
Other purification products
Phosphorylatedproteins
PhosphoproteinAffinity Column
Large-scale Small-scale
Magneticbead
separation
2015 4540353025 500 10Minutes
Enriched β-casein digest2,042kDa
3,140kDa
Clontech M 1 2 3
kDa
250 – 150 – 100 – 75 –
50 – 37 –
25 – 20 –
Inh
ibit
ion
(%
) 80
40
20
60
0
ProteoGuard
Competitor P
Glycoprotein Enrichment Resin & Detection Kit
• Flexible—enrichment resin can be used with gravity-flow or FPLC columns• Superior Performance—enrichment resin shows increased specific binding
of glycoproteins and reduced nonspecific binding• Specific—enrichment resin enriches for low- and high-abundance serum
glycoproteins
Phosphopeptide Enrichment—Small-Scale
• Rapid, reliable enrichment of phosphopeptides (30 minute protocol)• Available as magnetic beads or prepacked spin columns• Complete kits for microscale enrichment of phosphopeptides• Yields highly concentrated samples ideal for LC or MALDI analysis
Phosphoprotein Enrichment
• Large-Scale: Complete separation of phosphorylated and unphosphorylated proteins using gravity-flow affinity columns
• Small-Scale: Complete kit for magnetic bead-based microscale phosphoprotein enrichment from ANY cell or tissue samples (30 minute protocol)
• Percentage of phosphorylated proteins in final eluate: 14–17% (depends on cell type)
• Ideal for cell signaling studies (no radioactivity) or 2D-PAGE
ProteoGuard—Protease Inhibitor Cocktail
• Better protease inhibition than the leading competitor• More flexible than tablets• Optimized mix of five different inhibitors
Product InformationBrand Product Size Cat.No
Clontech HA-Tag Polyclonal Antibody 100 µg 631207
Clontech c-Myc Monoclonal Antibody 200 µg 631206
Clontech c-Myc Monoclonal Antibody-Agarose Beads 1ml 631208
O T H E R P R O T E I N P U R I F I C A T I O N
26
Thiophilic Antibody Purification Resins
• Purification at neutral pH—avoid antibody aggregates• High capacity—20–25 mg Ab/ml resin• Broad selectivity—purify IgY, IgM, IgE, and scAb• Yields highly stable purified antibodies• Reusable resin
Mag-Trypsin Immobilized Magnetic Trypsin
• Rapid and efficient protein digestion for mass spectrometry (MS) applications• Flexible—use the right amount of Mag-Trypsin for your application• Eliminates trypsin contamination for downstream applications
-0.05
0.250.200.150.1
0.050
0 5045403530252015105
UV
ab
sorb
ance
(21
5 n
m)
Minutes
β-casein digested with Mag-Trypsin
Filter/Cartridge
Addsalt
Load Wash
Elute in neutral buffer(pH 7.0)
Other purification products (continued...)
Product InformationBrand Product Size Cat.No
Clontech ProteoGuard EDTA-Free Protease Inhibitor Cocktail 10 x 100 µl 635673
Clontech Phosphoprotein Enrichment Kit 6 preps 635624
Clontech Phosphoprotein Enrichment Starter Kit 1 purification 635666
Clontech Phosphoprotein Kit—Buffer A 500 ml 635626
Clontech TALON® PMAC Magnetic Phospho Enrichment Kit1 each 635641
Clontech Phosphopeptide Enrichment Spin Columns 25 columns 635634
Clontech Phosphopeptide Enrichment Buffer Kit each 635635
Clontech Magnetic Phosphopeptide Enrichment Kit2 each 635643
Clontech Mag-Trypsin 5 ml 635646
Clontech Glycoprotein Western Detection Kit 20 rxns 635648
Clontech Glycoprotein Enrichment Resin 10 ml 635647
Clontech Antibody Purification—Thiophillic Uniflow Resin 100 ml 635614
Clontech Antibody Purification—Thiophillic Superflow Resin 10 ml100 ml
635616635617
O T H E R P R O T E I N P U R I F I C A T I O N
27
Takara’s Protein Sequencing and Analysis ProductsTakara offers a wide variety of Protein Fragmentation products as well as N-terminal deblocking and sequence determination and C-terminal sequence determination products.
Product Name Application DescriptionN-terminal and C-terminal analysis
Pfu Aminopeptidase I• Liberates N-terminal amino acids up to
X-Pro from proteins and peptides
Pfu Aminopeptidase I is a thermostable exo-type aminopep-tidase, isolated from Pyrococcus furiosus and produced as a recombinant protein, which liberates the N-terminal amino acid from proteins and peptides.
Pfu Pyroglutamate Aminopeptidase
• Removal of pyroglutamic acids from the N-terminal of proteins and peptides
• Deblocking of N-terminal pyroglutamates of proteins and peptides for sequence analysis using Edman degradation
Pfu Pyroglutamate Aminopeptidase liberates the N-terminal pyroglutamic acid from proteins and peptides. This enzyme may work well with some intact, non-denatured proteins and the denaturation step may be unnecessary in these instances.
Pfu Methionine Aminopeptidase • Liberates the N-terminal methionine
residues from proteins and peptides
Pfu Methionine Aminopeptidase specifically liberates only the N-terminal methionine residue from Met-X-Y when X is Ala, Gly, Ser, Thr, Pro or Val. This enzyme does not liber-ate the N-terminal Met when the N-terminus sequence is Met-Met-Y or Met-Met-Met-Y. It is not active toward formyl-methionine.
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP)
• N-Terminal deblocking
• N-terminal sequence analysis of blocked proteins or peptides
Pfu N-acetyl Deblocking Aminopeptidase (Ac-DAP) is a unique exo-type aminopeptidase that first liberates block-ing groups, such as formyl, acetyl, and myristyl, and then releases the first and subsequent amino acids from proteins and peptides until it reaches the first X-Pro bond.
Fragmentation of Proteins
Arginylendopeptidase• Fragmentation of proteins and pep-
tides required from primary structure analysis
Arginylendopeptidase cleaves peptide bonds at the carboxyl side of arginine residues found in pro teins and pep tides. Arginylendopeptidase is also known as mouse submaxillary pro tease D or as mouse EGF binding protein C.
Asparaginylendopeptidase• Fragmentation of proteins and peptides
required for primary structure analysis
Asparaginylendopeptidase specifically cleaves peptide bonds on the carboxyl side of asparagine residues found in proteins and peptides. Glycosylated asparagine residues are not cleaved.
Endoproteinase Asp-N• Fragmentation of proteins and peptides
required for primary structure analysis
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.
Pfu Protease S• Fragmentation of proteins and peptides
required for primary structure analysis
Pfu Protease S is an endo-type serine protease with broad recognition of native and denatured proteins. Cleavage occurs mainly on the carboxy side of peptide bonds of hydrophobic amino acid residues.
Protease Inhibitor
Calpastatin • Calpain protease inhibitor
Calpastatin is an endogenous protease in hib i tor that acts specifically on calpain calcium-dependent cysteine pro tease. It consists of four repetitive sequences of 120 to 140 amino acid residues (domains I, II, and IV), and an N-ter mi nal non-homologous sequence (L). The prod uct consists of highly purified recombinant human calpastatin domain I .
The fragmentation products are used for analysis of the primary structure of proteins and peptides.
Product InformationBrand Product Size Cat.No
Takara Pfu Pyroglutamate Aminopeptidase 10 mU 7334
Takara Pfu Methionine Aminopeptidase 20 mU 7335
Takara Pfu Aminopeptidase 0.5 mg 7336
Takara Pfu N-acetyl Deblocking Aminopeptidase 50 µg 7340
Takara Arginylendopeptidase 0.5 mg 7308
Takara Asparaginylendopeptidase 0.2 mU 7319
Takara Endoproteinase Asp-N 2 µg 7329
Takara Pfu Protease S 500 U 7339
Takara Calpastatin 3 mg 7316
P R O T E I N S E Q U E N C I N G A N D A N A L Y S I S
EUROPE
Takara Bio Europe, SAS
2, av. du Président Kennedy78100 St-Germain-en-LayeFrance
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Takara Bio, Inc. JAPAN
Takara Bio, Inc. USA A Takara Bio Company
Japan: +81.(0)77.543.724 Asia Pacific: +1.650.919.7300United States/Canada: +1.800.662.2566
Notice to Purchaser. Your use of these products and technologies is subject to compliance with any applicable licensing requirements described on the product’s web page at http://www.takarabio.com. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. Unless otherwise specified, other trade names are also the trademarks or registered trademarks of various companies.
For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Takara, the Takara logo, PrimeSTAR are trademarks of Takara Bio USA, Inc. Clontech, the Clontech logo, Tet-On, Tet-Express, TALON, In-Fusion are trademarks of Takara Bio USA, Inc. All other marks are the property of their respective owners. Certain trademarks may not be registered in all juridictions. ©2017 Takara Bio Europe.
ZZXBRTBE0317 EUThis brochure is printed on 60% recycled paper.
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