Alkaline Phosphatase. for the Diagnostics and Life Sciences Industry

Transcription

1 Alkaline Phosphatase for the Diagnostics and Life Sciences Industry

2 Table of Contents Your Perfect Partner 3 Alkaline Phosphatase (AP) Product Overview 4 5 Characteristics of Recombinant and Native Alkaline Phosphatase 6 Features of Recombinant AP Conjugates 7 Recombinant AP Mutein: Inactive AP for Interference Elimination 8 Recombinant Alkaline Phosphatase for Molecular Diagnostics 9 Chromatography columns for the large scale production of biosubstances References 10 Products are for further processing only.

3 Your Perfect Partner Roche Custom Biotech is ready to serve you in a long-term relationship as a quality-conscious, reliable partner for raw material supply. Our experience in immunology and molecular diagnostics supports your diagnostic kit manufacturing, comprehensively and professionally, from the beginning of assay development to commercialization. Peeler centrifuge with automated drawdown for sealed separation of solids Chromatography equipment for large-scale production of enzymes: Fraction containers 3

4 Alkaline Phosphatase (AP) Product Overview The AP portfolio of Roche Custom Biotech comprises two AP product classes: - Native AP from New Zealand bovine intestine (BSE-free) - Recombinant AP produced in the yeast Pichia pastoris The high quality of both of these product groups serves all your needs for immunology and molecular diagnostics. These AP products are the perfect building blocks for stable and reproducible AP conjugates. Recombinant Alkaline Phosphatase, from Pichia pastoris for immuno-diagnostics: --EIA Grade, highly active --EIA Grade, highly active, carbohydrate-reduced --AP Mutein, inactive AP Alkaline Phosphatase, bovine intestine, EIA Grade Recombinant Alkaline Phosphatase, Molecular Biology Grade Substrates for the detection of AP conjugates: --pnpp for ELISAs --BCIP for blots and immunohistochemistry and immunocytochemistry AP Mutein, the inactive variant of the recombinant highly active AP, is also available from Roche Custom Biotech for applications involving AP interference elimination. Properties of Alkaline Phosphatase Nomenclature Orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC Subunits Molecular weight (MW) Inhibitors Homodimer (Zn 2+ is essential for activity) 56 kd (by SDS-PAGE, monomer) Activators Mg 2+, Co 2+, Mn 2+ ph Optimum ph Optimum Specificity Inorganic phosphate, metal chelating agents, divalent heavy metal ions (e.g., Be 2+, Zn 2+ ), many amino acids (e.g., L-phenylalanine, L-tryptophane, L-cysteine), iodoacetamide 9.8 (activity) 8.0 (stability) Alkaline Phosphatase catalyzes the hydrolysis of numerous phosphate esters, such as esters of primary and secondary alcohols, saccharides, cyclic alcohols, phenols and amines. Phosphodiesters do not react. The enzyme hydrolyzes inorganic pyrophosphate. The kinetic properties of the enzyme depend on many factors, such as purity of enzyme, concentration of enzyme in the assay, buffer, ph etc. 4

5 Native Alkaline Phosphatase, EIA Grade AP, EIA Grade is specially developed as a raw material for producing stable and highly repro ducible antibody- and antigen-enzyme conjugates. These AP conjugates are ideal for enzyme immunoassays, providing a wide range of detection options using colorimetric, fluorimetric or luminometric substrates. Native AP, EIA Grade is: the standard for alkaline phosphatase conjugates from bovine intestine sourced from New Zealand, a country acknowledged as being BSE-free Model 3D structure of the homo dimer of recombinant highly active AP KK AP subunits Catalytic active Ser 92: AP Mutein recombinant is created by the single point mutation Ser 92 Ala 92 which yields an inactive enzyme. Asn residues: Potential glycosylation sites Lys residues Recombinant Alkaline Phosphatase Recombinant highly active AP is the ideal alternative to conventional AP products derived from bovine intestine. No animal-derived components are used in the production process. Therefore the risk of Bovine Spongiform Encephalopathy (BSE) or other infections caused by animals is eliminated. Recombinant AP of Roche Custom Biotech: uses the yeast, Pichia pastoris, as expression system. has a clearly defined molecular structure. has a high specific activity: 7000 U/mg. is also available in a carbohydrate-reduced (CR) quality. Highly active recombinant AP comprises fewer isoenzymes and has just slightly different N-glycosylation compared to native AP. This N-glycosylation difference is due to production in Pichia pastoris, which produces a slightly higher mannose branching pattern. An optional proprietary deglycosylation digests Recombinant AP s mannose branching pattern. This treatment produces a highly active, carbo hydratereduced, recombinant AP with a carbo hydrate ratio comparable to that of native AP. Model for N-glycosylation Native AP Average Carbohydrates: 10% M Asn GN GN M GN M GN GN Recombinant AP 16 22% Asn GN GN M M [M] 0-2 M [M] 0-2 Recombinant AP, CR 6% Asn GN M Asn GN GN M GN Asn GN GN M Asn GN M GN GN For additional information please refer to references 2) and 3). M M [M] 0-2 [M] 0-2 Asn: Asparagine GN: GlcNAc M: Mannose 5

6 Characteristics of Recombinant and Native Alkaline Phosphatase Physical data Native AP, EIA Grade Recombinant AP, highly active Recombinant AP, highly active, CR Catalog Number IEP (IEF, CE) MALDI-TOF MS Total molecular mass Molecular mass protein Molecular mass carbohydrates Accessible N-glycosylation sites Branching type 116 kd 104 kd (= 90%) 12 kd (= 10%) 2/subunit high branched type (hybrid type) GlcNAc, Mannose, no NeuAc detected 124 ± 10 kd 104 kd (= 84%) 20 ± 10 kd (16 ± 6%) 2/subunit higher branched type (hybrid type) GlcNAc, Mannose, no NeuAc detected 111 kd 104 kd (= 94%) 6.5 kd (= 6%) 2/subunit reduced branched type GlcNAc, Mannose, no NeuAc detected O-glycosylation sites Not detected Not detected Not detected Number of isoenzymes present based on protein 7 (IEF-2D) 5 (IEF-2D) 3 (MS) 1 (MS) 1 (MS) 1 (MS) Specifications Native AP, EIA Grade Recombinant AP, highly active Recombinant AP, highly active, CR Catalog Number Appearance Clear colorless solution in NaCl, 3 mol/l; ZnCl 2, 0.1 mmol/l; TEA, 30 mmol/l; MgCl 2, 1 mmol/l Clear colorless solution in NaCl, 3 mol/l; ZnCl 2, 0.1 mmol/l; TEA, 30 mmol/l; MgCl 2, 5 mmol/l Clear colorless solution in NaCl, 3 mol/l; ZnCl 2, 0.1 mmol/l; TEA, 30 mmol/l; MgCl 2, 5 mmol/l ph Value Protein (A 280 : 1 mg/ml = 1; against H 2 O) 10 mg/ml 20 ± 1 mg/ml 20 ± 1 mg/ml Specific activity (37 C; pnpp) 3000 U/mg 7000 U/mg 7000 U/mg Purity (HPLC) 95% 95% 95% Amino groups 8 16 moles/mole 5 13 moles/mole 5 13 moles/mole Carbohydrates No limit No limit 7% Stability +2 to +8 C 15 months +2 to +8 C 12 months +2 to +8 C 12 months 6

7 Features of Recombinant AP Conjugates Conjugation experiments show that highly active, recombinant Alkaline Phosphatase, EIA Grade is the ideal replacement for conventional alkaline phosphatase products. Recombinant AP conjugates show: similar chromatographic profiles compared to conventional AP products high reproducibility and lot-to-lot consistency of conjugates (see stability data below) high sensitivity in ELISAs Stability of < DIG > -AP conjugates with rec. AP, highly active, EIA Grade (2 lots) and bovine AP, highly active, EIA Grade (1 lot) at 4 C at 35 C Recovery of enzyme activity [%] Recovery of enzyme activity [%] weeks weeks Conjugation of AP with PAB<DIG>S-Fab(IS) via Nakane (AP ox ): Recombinant, highly active AP displays high stability, which is nearly identical to the native enzyme. In Roche's model system, Fab<DIG>-AP conjugates exhibit good repro ducibility and lot-to-lot consistency. 7

8 Recombinant AP Mutein: Inactive AP for Interference Elimination Recombinant AP Mutein is the inactive form of the highly active Recombinant AP. AP Mutein eliminates interference directed against the active AP. The inactivating moiety in AP Mutein is a single point mutation located in the AP active site. Recombinant AP Mutein is the optimal choice for reducing AP-related interference in your applications. Recombinant AP Mutein: is equivalent to highly active Recombinant AP with the exception of a single inactivating point mutation. is produced using the same initial production steps as the active enzyme in Pichia pastoris. production involves the same deglycosylation step as established for highly active AP, CR. has a specific activity of 10 U/mg. is provided in EIA Grade quality. Specifications Recombinant AP Mutein Catalog Number Appearance ph Value White to yellowish lyophilizate Protein (A 280 ; 1 mg/ml = 1; against H 2 0) 0.2 mg protein/mg Iyophilizate Specific activity (37 C; pnpp) 10 U/mg protein Stability +2 to +8 C 24 months 8

9 Recombinant Alkaline Phosphatase for Molecular Diagnostics Recombinant Alkaline Phosphatase, Molecular Biology Grade meets all your needs for applications in molecular biology and diagnostics. Additional production steps ensure the removal of DNases, RNases, nicking activity and exonucleases, which can lead to the degradation of DNA and RNA. In addition to its consistent quality and stability, the major advantage of Recombinant Alkaline Phosphatase, Molecular Biology Grade, is its simple inactivation by heating at +75 C for 5 minutes. Recombinant AP, Mol. Biol. Grade, is especially suitable for dephosphorylation of 5' phosphorylated ends of DNA and RNA. Dephosphorylated vector DNA is prevented from self-annealing prior to the insertion of DNA fragments. The special quality of Recombinant AP, Molecular Biology Grade avoids the degradation of probeenzyme conjugates used to detect amplified and non-amplified DNA/RNA in probe detection assays. The entire workflow, including restriction enzyme digestion, dephosphorylation, enzyme inactivation, ligation or 5' end-labeling, can be performed in a single tube. Specifications Recombinant AP, Mol Biol Grade 1 U/µl Recombinant AP, Mol Biol Grade 20 U/µl Catalog Number Appearance Clear colorless solution in Tris-HCI, 25 mmol/l; MgCl 2, 1 mmol/l; ZnCl2, 0.1 mmol/l; glycerol, 50% (v/v); ph approximately 7.6 at 4 C Volume activity 1 x 10 3 U/ml 20 x 10 3 U/ml Specific activity 5 x 10 3 U/mg 5 x 10 3 U/mg Endonucleases Not detectable ( 100 U/4 h/37 C) Not detectable ( 100 U/4 h/37 C) Exonucleases Not detectable ( 60 U/4 h/37 C) Not detectable ( 60 U/4 h/37 C) RNases Not detectable ( 100 U/1 h/37 C) Not detectable ( 100 U/1 h/37 C) Nicking activity Not detectable ( 100 U/4 h/37 C) Not detectable ( 100 U/4 h/37 C) 5'-Labeling Typical incorporation rate 30% Typical incorporation rate 30% Stability -15 to -25 C 24 months -15 to -25 C 24 months 9

10 References 1) Genetic Complexity, Structure and Characterisation of Highly Active Bovine Intestinal Alkaline Phosphatases, JBC, 273, 36, (1998), Th. Manes, M.F. Hoylaerts, R. Mueller, F. Lottspeich, W. Hoelke, J.L. Millán 2) Glycosylation of Pichia Pastoris derived Proteins, Biotechnol. Appl. Biochem. 30, (1999), R.K. Bretthauer, F.J. Castellino 3) Heterologous Protein Expression in the Methylotropic Yeast Pichia Pastoris FEMS Microbiology Reviews 24, (2000), I. Ceveghino, I. Gregg 4) Heterologous Protein Production in Methylotropic Yeasts, Appl. Microbiol. Biotechnol. 54, (2000), G. Gellissen 5) Detection of attograms of antigen by a high-sensitivity enzyme-linked immunoabsorbent assay (HS-ELISA) using a fluorogenic substrate, J. Immunol. Meth. 38, 125 (1980), A. Shalev, A. H. Greenberg and P. McAlpine 6) Which of the commonly used marker enzymes gives the best results in colorimetric and fluorometric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or b-galactosidase? J. Immunol. Meth. 79, 27 (1985), B. Porstmann, T. Porstmann, E. Nugel and U. Evers Products are for further processing only. 10

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12 Published by Roche Diagnostics GmbH Sandhofer Straße Mannheim Germany 2010 Roche Diagnostics. All rights reserved Your Roche Custom Biotech Customer Service Customers in Europe, Middle East, Africa, and Latin America: Roche Diagnostics Deutschland GmbH Sandhofer Straße Mannheim, Germany Phone Fax Customers in the United States: Roche Diagnostics Corporation Roche Applied Science 9115 Hague Road P.O. Box Indianapolis, IN , USA Phone , ext (toll-free) Fax Customers in Canada: Roche Diagnostics 201, Boulevard Armand-Frappier HV 4A2 Laval, Québec, Canada Phone Fax Customers in Japan: Roche Diagnostics K.K. Roche Custom Biotech Applied Science 6-1, Shiba 2-chome Minato-ku, Tokyo , Japan Phone Fax Customers in Asia Pacific: Roche Diagnostics Asia Pacific Pte. Ltd. Regional Sales and Market Development 298 Tiong Bahru Road # 16-01/06 Central Plaza Singapore, Phone Fax custombiotech.roche.com