Reduction of Protein Degradation by Use of Protease-Deficient Mutants in Cell-Free Protein Synthesis System of Escherichia coli
スポンサーリンク
概要
- 論文の詳細を見る
In an Escherichia coli in vitro transcription/translation system, the degradation of produced proteins is often caused by endogenous proteases from E. coli extracts. To reduce the extent of this degradation, several extracts were prepared from E. coil mutants that genetically lacked DegP, OmpT, or Lou proteases. Then, these extracts were used with ^<14>C-leucine in a system for synthesizing single-chain Fv against gp120 (anti-gp120), and phospholipase D (PLD) of Streptomyces antibioticus. The proteins synthesized in vitro were analyzed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE) and autoradiography. The use of extracts from mutants that were deficient in the structural genes encoding OmpT and Lon markedly repressed the degradation of anti-gp120. Similarly, extracts from degP- and ompT-deleted mutants were able to significantly stabilize in vitro-synthesized PLD, which otherwise disappeared within 30 mm. Such protease-deficient mutants were suggested to be useful for preventing the degradation of heterologous proteins in in vitro systems.
- 社団法人日本生物工学会の論文
- 2002-02-25
著者
-
Nakano Hideo
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya Universit
-
YAMANE TSUNEO
Laboratory of Molecular Biotechnology, Division of Molecular and Cellular Mechanisms, graduate Schoo
-
Iwasaki Yugo
Laboratory of Molecular Biotechnology, Department of Bioengineering Sciences, Graduate School of Bio
-
Iwasaki Y
Lab. Of Molecular Biotechnology Dep. Of Bioengineering Sciences Graduate School Of Bioagricultural S
-
Iwasaki Yugo
Laboratory Of Molecular Biotechnology Department Of Bioengineering Sciences Graduate School Of Bioag
-
Iwasaki Yugo
Laboratory Of Molecular Biotechnology Graduate School Of Bio- And Agro-sciences Nagoya University
-
Iwasaki Yugo
Department Of Applied Biological Sciences Faculty Of Agriculture Nagoya University
-
Yamane T
Laboratory Of Molecular Biotechnology Graduate School Of Bioagricultural Sciences Nagoya University:
-
Yamane Tsuneo
Laboraotory Of Bioreaction Engineering Department Of Food Science And Technology Faculty Of Agricult
-
Yamane Tsuneo
Laboratory Of Bioreaction Engineering Department Of Food Science And Technology School Of Agricultur
-
Yamane T
Laboratory Of Molecular Biotechnology Graduate School Of Biological & Agricultural Sciences Nago
-
Yamane T
Laboratory Of Bioreaction Engineering Department Of Food Science And Technology School Of Agricultur
-
JIANG XIUPING
Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nago
-
Ichihara Shigeyuki
Laboratory Of Alicrobiology School Of Agriculture Nagoya University
-
Ichihara Shigeyuki
Department Of Applied Biological Science Faculty Of Agriculture Nagoya University
-
Ichihara Shigeyuki
Department Of Agricultural Sciences Meijo University
-
Nakano Hideo
Laboratory Of Molecular Biotechnology Graduate School Of Bioagricultural Sciences Nagoya University
-
Jiang Xiuping
Laboratory Of Molecular Biotechnology Graduate School Of Biological And Agricultural Sciences Nagoya
-
Yabuki Takashi
Protein Research Group Riken Genomic Sciences Center
-
OOHIRA KAZUYO
Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nago
-
Yamane Tsuneo
Dept. Of Food Science And Technology Faculty Of Agriculture Nagoya University
-
Oohira Kazuyo
Laboratory Of Molecular Biotechnology Graduate School Of Biological And Agricultural Sciences Nagoya
-
Yamane Tsuneo
Laboratory Of Molecular Biotechnology Graduate School Of Bio- And Agro-sciences Nagoya University
-
Nakano Hideo
Laboratory Of Molecular Biotechnology Department Of Bioengineering Sciences Graduate School Of Bioag
-
Nakano Hideo
Department Of Bioengineering Sciences Graduate School Of Bioagricultural Sciences Nagoya University
関連論文
- High-throughput screening of DNA binding sites for transcription factor AmyR from Aspergillus nidulans using DNA beads display system(GENETICS, MOLECULAR BIOLOGY, AND GENE ENGINEERING)
- Directed evolution of angiotensin II-inhibiting peptides using a microbead display(METHODS)
- In vitro generation of anti-hepatitis B monoclonal antibodies from a single plasma cell using single-cell RT-PCR and cell-free protein synthesis(MEDICAL BIOTECHNOLOGY)
- Stabilization of Affinity-Tagged Recombinant Protein during/after Its Production in a Cell-Free System Using Wheat-Germ Extract
- 2Ea06 相互作用ドメイン過剰発現による出芽酵母オートファジー阻害機構の解析(タンパク質工学,一般講演)
- 2Dp01 無細胞蛋白質合成系を用いたセルラーゼの進化分子工学(酵素学・酵素工学,一般講演)
- 1Ep21 新規な定量的高速スクリーニング法を用いたヒトMad2蛋白質結合ペプチドの創製(タンパク質工学,一般講演)
- 2Da05 酵素によるホスファチジルイノシトール異性体の組成分析(酵素学・酵素工学,一般講演)
- 2Ep08 一細胞RT-PCRと無細胞蛋白質合成による抗インフルエンザウイルスモノクローナル抗体の取得(抗体工学,一般講演)
- 2Dp21 ビーズディスプレイ法を用いたリパーゼのハイスループットスクリーニング法の開発(酵素学・酵素工学,一般講演)
- 2Dp15 エマルジョン培養法による分泌酵素産生菌の選択的濃縮(酵素学・酵素工学,一般講演)
- 2Da06 改変型ホスホリパーゼDを用いた環状二級アルコールに対するリン脂質化(酵素学・酵素工学,一般講演)
- 2Da04 放線菌由来ホスホリパーゼDの耐熱化(酵素学・酵素工学,一般講演)
- 2Cp14 DNAのビーズディスプレイ法を用いたDNA-酵母転写因子相互作用検出システム(核酸工学・糖鎖工学・ペプチド工学,一般講演)
- エマルジョンPCRが拓く分子間相互作用ハイスループットスクリーニング(分子機能を拓く)
- 特集によせて(分子機能を拓く)
- 2Fa03 1型および3型ホスファチジルイノシトールを優先的に合成する変異型ホスホリパーゼDの機能解析(酵素学,酵素工学,タンパク質工学,一般講演)
- 2Fa04 改変型ホスホリパーゼDを用いたホスファチジルイノシトールの合成(酵素学,酵素工学,タンパク質工学,一般講演)
- 3Fa12 相互作用ドメイン過剰発現による出芽酵母オートファジー関連蛋白質間相互作用阻害の生化学的解析(ペプチド工学・プロテオーム,一般講演)
- 3Fa14 半定量的酵母2ハイブリッド系を用いた相互作用蛋白質の高速スクリーニング(ペプチド工学・プロテオーム,一般講演)
- 2Ga14 一細胞PCRと無細胞蛋白質合成系による抗ガングリオシド抗体の取得(酵素学,酵素工学,タンパク質工学,一般講演)
- 2Da07 DNAのビーズディスプレイ法を用いたDNA : 転写因子間相互作用検出法(遺伝子工学,核酸工学,一般講演)
- 2C10-1 変異型ホスホリパーゼDにより合成されたホスファチジルイノシトールの異性体解析(酵素学・酵素工学・タンパク質工学,一般講演)
- Directed evolution of angiotensin II-inhibiting peptides using a microbead display
- Synthesis of Phospholipids Containing Polyunsaturated Fatty Acids by Phospholipase A2-mediated Esterification with Food-compatible Reagents
- 1F11-5 位置特異的アミノ酸飽和変異による2-デオキシリボース-5-リン酸アルドラーゼの基質特異性改変(酵素学,酵素工学,タンパク質工学,一般講演)
- 1J16-5 特定蛋白質間相互作用の選択的破壊法の開発(食品科学,食品工学/有機化学,高分子化学/ペプチド工学,プロテオーム,一般講演)
- 2D13-5 進化工学的手法を用いた出芽酵母キネトコアDam1複合体のサブユニット間相互作用領域の同定(ペプチド工学・プロテオーム,生合成・天然物化学,一般講演)
- In Vitro Construction and Screening of a Burkholderia cepacia Lipase Library Using Single-Molecule PCR and Cell-Free Protein Synthesis(METHODS)
- Single-Step Single-Molecule PCR or DNA with a Homo-Priming Sequence Using a Single Primer and Hot-Startable DNA Polymerase
- 1B17-3 A Novel Compact Disk-like Microfluidic Device with Micropillars for Enzyme-linked Immunosorbent Assay
- 755 Monitoring of lipase-catalyzed transesterification between eicosapentaenoic acid ethyl ester and tricaprylin by silver ion high performance liquid chromatography and high temperature gas chromatography
- Decreasing Accumulation of Acetate in a Rich Medium by Escherichia coli on Introduction of Genes on a Multicopy Plasmid
- P-405 SITE-SPECIFIC FUNCTIONALIZATION OF PROTEINS BY ORGANOPALLADIUM REACTIONS
- Improvements in the Cell-Free Production of Functional Antibodies Using Cell Extract from Protease-Deficient Escherichia coli Mutant (Methods)
- EP-P19 Beads display : Genotype-phenotype coupling screening of DNA molecules displayed on microbeads by using emulsion PCR and in vitro protein synthesis(Section VII Enzyme and Protein Engineering)
- Construction of Pta-Ack Pathway Deletion Mutants of Escherichia coli and Characteristic Growth Profiles of the Mutants in a Rich Medium
- Generation of Monoclonal Antibodies Using Simplified Single-Cell Reverse Transcription-Polymerase Chain Reaction and Cell-Free Protein Synthesis(METHODS)
- Modulator-Mediated Synthesis of Active Lipase of Pseudomonas sp.109 by Escherichia coli Cell-Free Coupled Transcription/Translation System
- Accumulation of Translational Inhibitor durig Multi-Hour Cell-Free Protein Synthesis Reaction Using Rabbit Reticulocyte Lysate
- High Speed Polymerase Chain Reaction in Constant Flow
- 448 In vitro analysis of the roles of a disulfide bridge and a calcium binding site in Pseudomonas sp. KWI-56 lipase using PCR and Escherichia coli coupled transcription/translation
- Increasing Productivity by Removing Choline in Conversion of Phosphatidylcholine to Phosphatidylserine by Phospholipase D
- 573 Increased Productivity by Removing Choline in Conversion of Phosphatidyl-choline to Phosphatidylserine by Phospolipase D.
- 515 Increasing Phosphatidylcholine Content of Lecithin by Phospholipase D
- 226 Enzymatic Conversion of Phosphatidylcholine to Phosphatidylethanolamine by Phospholipase D from Different Sources
- 327 Continuous Ethanol Fermentation Using Flocculent Yeast Entrapped in Horizontal Parallel Flow Bioreactor System
- Phenomenological Background and Some Preliminary Trials of Automated Substrate Supply in pH-Stat Modal Fed-Batch Culture Using a Setpoint of High Limit
- L-Serine Production from Methanol and Glycine with an Immobilized Methylotroph
- Economic Significance of Increasing Cell Mass Concentration for Metabolite Production : A Theoretical Study
- Effect and Control of Glucose Feeding on Bacitracin Production by Fed-Batch Culture of Bacillus licheniformis
- Production of L-Serine from Methanol and Glycine by Resting Cells of a Methylotroph under Automatically Controlled Conditions
- Control of Carbon Source Supply and Dissolved Oxygen by Use of Carbon Dioxide Concentration of Exhaust Gas in Fed-Batch Culture
- Detection and Automatic Control of Ammonium Ion Concentration in Microbial Culture with an Ammonium Ion Selective Electrode
- 565 Charcteristics of Batch Culture of a Recombinant ___- ___- Producing a Foreign Esterase
- Comprison of Cell Productivities among Fed-Batch, Repeated Fed-Batch and Continuous Cultures at High Cell Concentration
- Inhibition and Inactivation of Lipase by Fat Peroxide in the Course of Batch and Continuous Glycerolyses of Fat by Lipase(Biological Chemistry)
- Selective Hydrolysis of Fish Oil by Lipase to Concentrate n-3 Polyunsaturated Fatty Acids(Biological Chemistry)
- Purification and Some Characteristics of Extracellular Lipase from Fusarium oxysporum f.sp.lini
- In Vitro Selection of DNA Binding Sites for Transcription Factor, PhaR, from Paracoccus denitrificans Using Genetic Library on Microbeads and Flow Cytometry(GENETICS, MOLECULAR BIOLOGY, AND GENE ENGINEERING)
- 1F14-1 Enzymatic preparation of enantiomerically pure 2,3-diacylglycerol
- 2J10-5 Direct separation of regio- and enantiomeric isomers of diacylglycerols by a tandem column high-performance liquid chromatography
- Expression Profiles of Polyhydroxyalkanoate Synthesis-Related Genes in Paracoccus denitrificans
- Cloning and in Vitro and in Vivo Expression of Plant Glutathione S-Transferase Zeta Class Genes(GENETICS,MOLECULAR BIOLOGY,AND GENE ENGINEERING)
- Reduction of Protein Degradation by Use of Protease-Deficient Mutants in Cell-Free Protein Synthesis System of Escherichia coli
- Dosage Effect of Minor Arginyl- and Isoleucy1-tRNAs on Protein Synthesis in an Escherichia coli In Vitro Coupled Transcription/Translation System
- Importance of Disulfide Bridge Formation on Folding of Phospholipase D from Streptomyces antibioticus
- Continuous Production of Phospholipase D by Streptomyces lydicus D-121 Immobilized with Cross-Linked Chitosan Beads
- Acquisition of Data from On-Line Laser Turbidimeter and Calulation of Some Kinetic Variables in Computer-Coupled Automated Fed-Batch Culture
- Extracellular Production of Phospholipase D of Streptomyces antibioticus Using Recombinat Escherichia coli
- Prolonged Cell-free Protein Synthesis in a Batch System Using Wheat Germ Extract
- Crystal Structure of Bovine Trypsin and Wheat Germ Trypsin Inhibitor (I-2b) Complex (2 : 1) at 2.3 A Resolution
- Phosphatidylglycerol Synthesis by Phospholipase D in a Microporous Membrane Bioreactor : Studies on Enzymatic Conversion of Phospholipids (I)
- Purification and Properties of Phospholipase D from Streptomyces antibioticus
- 107 Effective Extracellular Production of Bacillus stearothermophilus Esterase by pH-stat modal Fed-batch Culture of Recombinant Bacillus brevis
- An Increased Rate of Cell-free Protein Synthesis by Condensing Wheat-germ Extract with Ultrafiltration Membranes
- Immobilization of Yeast Cells with Cross-Linked Chitosan Beads
- Nonadditive Effects of Double Mutations at the Flexible Loops, Glycine-67 and Glycine-121, of Escherichia coli Dihydrofolate Reductase on Its Stability and Function
- Effects of Point Mutations at the Flexible Loop Glycine-67 of Escherichia coli Dihydrofolate Reductase on Its Stability and Function
- Site-specific incorporation of 4-lodo-L-phenylalanine through opal suppression
- Apoptotic Cell Death in the Fission Yeast Schizosaccharomyces pombe Induced by Valproic Acid and Its Extreme Susceptibility to pH Change
- Three Automated Feeding Strategies of Natural Complex Nutrients Utilizing On-Line Turbidity Values in Fed-Batch Culture : A Case Study on the Cultivation of a Marine Microorganism
- Interesterification of Fats and Oils by Immobilized Fungus at Constant Water Concentration
- Kinetic Studies on the Interesterification of Oils and Fats Using Dried Cells of Fungus
- Screening for Fungi That Have High Lipolytic and Acidolytic Activities in Biomass Support Particles
- Avoidance of Solidification of Sesame Oil at Low Temperature by Self-interesterification with Immobilized Lipase
- Purification and Characterization of Nicotinic Acid Dehydrogenase from Pseudomonas fluorescens TN5
- Microbial Production of 6-Hydroxynicotinic Acid, an Important Building Block for the Synthesis of Modern Insecticides
- Production of 6-Hydroxynicotinic Acid from Nicotinic Acid by Resting Cells of Pseudomonas fluorescens TN5
- A Bacillus stearothermophilus Esterase Produced by a Recombinant Bacillus brevis Stabilized by Sulfhydryl Compounds
- 316. Purification and Characterization of a Thermostable Bacillus stearothermophilus Esterase Excreted from a Recombinant Bacillus brevis
- Purification and Properties of a Thermostable Esterase of Bacillus stearothermophilus Produced by Recombinant Bacillus brevis
- Replica Technique for Screening Poly(3-Hydroxyalkanoic Acid)-Producing Bacteria by Nile Blue Staining
- Synthesis of phosphatidylinositols having various inositol stereoisomers by engineered phospholipase D(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
- Apoptotic Cell Death in the Fission Yeast Schizosaccharomyces pombe Induced by Valproic Acid and Its Extreme Susceptibility to pH Change
- Emulsion culture : A miniaturized library screening system based on micro-droplets in an emulsified medium(METHODS)
- High-throughput screening method for promoter activity using bead display and a ligase ribozyme(METHODS)
- Some Characteristics of Continuous Glyceride Synthesis by Lipase in a Microporous Hydrophobic Membrane Bioreactor
- Continuous Hydrolysis of Olive Oil by Lipase in Microporous Hydrophobic Hollow Fiber Bioreactor
- High-throughput screening method for promoter activity using bead display and a ligase ribozyme