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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 Takara Bio Europe • www.takara-bio.eu • [email protected] 04_EU_cat09_PRORES_FINAL_v00.indd 1 4.1 3/11/2009 1:25:09 PM 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 04_EU_cat09_PRORES_FINAL_v00.indd 2 Takara Bio Europe • www.takara-bio.eu • [email protected] 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] 04_EU_cat09_PRORES_FINAL_v00.indd 3 4.3 3/11/2009 1:25:12 PM 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 04_EU_cat09_PRORES_FINAL_v00.indd 4 Takara Bio Europe • www.takara-bio.eu • [email protected] 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. Takara Bio Europe • www.takara-bio.eu • [email protected] 04_EU_cat09_PRORES_FINAL_v00.indd 5 PROTEIN RESEARCH pCold TF DNA 4.5 3/11/2009 1:25:14 PM 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 Takara Bio Europe • www.takara-bio.eu • [email protected] 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. Takara Bio Europe • www.takara-bio.eu • [email protected] 04_EU_cat09_PRORES_FINAL_v00.indd 7 4.7 3/11/2009 1:25:19 PM 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] 4.8 04_EU_cat09_PRORES_FINAL_v00.indd 8 Takara Bio Europe • www.takara-bio.eu • [email protected] 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 Takara Bio Europe • www.takara-bio.eu • [email protected] 04_EU_cat09_PRORES_FINAL_v00.indd 9 4.9 3/11/2009 1:25:22 PM 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 4.10 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] 04_EU_cat09_PRORES_FINAL_v00.indd 11 4.11 3/11/2009 1:25:28 PM 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 4.12 04_EU_cat09_PRORES_FINAL_v00.indd 12 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 4.13 3/11/2009 1:25:30 PM 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. 4.14 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. Takara Bio Europe • www.takara-bio.eu • [email protected] 04_EU_cat09_PRORES_FINAL_v00.indd 15 4.15 3/11/2009 1:25:31 PM 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 4.16 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) Takara Bio Europe • www.takara-bio.eu • [email protected] 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) Takara Bio Europe • www.takara-bio.eu • [email protected] 04_EU_cat09_PRORES_FINAL_v00.indd 17 4.17 3/11/2009 1:25:32 PM 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 4.18 04_EU_cat09_PRORES_FINAL_v00.indd 18 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). Takara Bio Europe • www.takara-bio.eu • [email protected] 3/11/2009 1:25:32 PM