RESUMO
A novel phospholipase C gene (TtPLC) from Thielavia terrestris CAU709 was cloned and efficiently expressed in Pichia pastoris. The deduced protein sequence of TtPLC shared the highest identity of 33% with the characterized phospholipase C from Arabidopsis thaliana. The highest phospholipase C yield of 98, 970 U mL-1, with a protein concentration of 4.9 mg mL-1 was obtained by high-cell density fermentation in a 5-L fermentor. The recombinant enzyme (TtPLC) was purified to homogeneity with a recovery yield of 59.1% and a specific activity of 22, 910 U mg-1. TtPLC was most active at pH 6.5 and 55 °C, respectively. It was stable within the pH range of 4.5-8.0 and up to 45 °C. The enzyme exhibited excellent stability in different surfactants and organic solvents, including Tween 20 (147.6%), Tween 40 (180.6%), Tween 60 (205.4%), cyclohexane (160.0%), n-octane (178.2%), n-heptane (180.7%), acetone (187.5%) etc. The application of TtPLC in crude soybean oil degumming process significantly reduced the residual phosphorus content from 135.4 mg kg-1 to 7.9 mg kg-1 under the optimized conditions, which satisfied the requirement of environmental friendly physical refining process for oil refining industry. Therefore, TtPLC should be a good candidate in oil refining industry.
Assuntos
Biodegradação Ambiental , Expressão Gênica , Sordariales/enzimologia , Sordariales/genética , Fosfolipases Tipo C/genética , Sequência de Aminoácidos , Reatores Biológicos , Clonagem Molecular , Ativação Enzimática , Fermentação , Proteínas Recombinantes , Análise de Sequência de DNA , Especificidade por Substrato , Fosfolipases Tipo C/isolamento & purificação , Fosfolipases Tipo C/metabolismoRESUMO
The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247-370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringensl-lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin.
Assuntos
Anticorpos Antibacterianos/sangue , Toxinas Bacterianas , Proteínas de Ligação ao Cálcio , Proteínas Recombinantes , Fosfolipases Tipo C , Animais , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/isolamento & purificação , Vacinas Bacterianas , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/isolamento & purificação , Toxina da Cólera/genética , Clonagem Molecular , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Escherichia coli/genética , Camundongos , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Fosfolipases Tipo C/biossíntese , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/imunologia , Fosfolipases Tipo C/isolamento & purificação , Vacinação/métodosRESUMO
Objective: In this study, we constructed recombinant Kluyveromyces lactis strains to produce phospholipase C (PLC) of Bacillus cereus. The recombinant enzymes were purified and characterized. Methods: We cloned the PLC encoding gene bcplc of Bacillus cereus. And the amplified fragments were inserted into pKLAC1 to obtain expression plasmids. K. lactis harboring the above plasmids was cultivated to express PLC that was purified by HisTrapTM affinity chromatography and characterized. Results: PLC of B. cereus was cloned and expressed in K. lactis. The recombinant enzyme had shown activity of 19251 U/mg when using p-nitrophenyl phosphorycholine as substrate. Purified PLC exhibited optimum temperature at 80 °C and optimal pH at 9.0. The recombinant enzyme was stable below 40 °C and pH between 7.0 and 8.0. Cu2+ and Co2+ inhibited its activity whereas Zn2+, Mn2+, Ca2+ and Mg2+ stimulated its activity. Conclusion: It is the first time to express and characterize the PLC gene in K. lactis. These research results provide reference for the study of recombinant PLC.
Assuntos
Bacillus cereus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Expressão Gênica , Kluyveromyces/genética , Fosfolipases Tipo C/química , Fosfolipases Tipo C/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Kluyveromyces/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Temperatura , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce phosphate monoesters and diacylglycerol. It has many applications in the enzymatic degumming of plant oils. PLC Bc , a bacterial PLC from Bacillus cereus, is an optimal choice for this activity in terms of its wide substrate spectrum, high activity, and approved safety. Unfortunately, its large-scale production and reliable high-throughput screening of PLC Bc remain challenging. Herein, we summarize the research progress regarding PLC Bc with emphasis on the screening methods, expression systems, catalytic mechanisms and inhibitor of PLC Bc . This review hopefully will inspire new achievements in related areas, to promote the sustainable development of PLC Bc and its application.
Assuntos
Bacillus cereus/enzimologia , Inibidores Enzimáticos/farmacologia , Fosfolipases Tipo C/biossíntese , Bacillus cereus/química , Bacillus cereus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Especificidade por Substrato , Fosfolipases Tipo C/química , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/isolamento & purificaçãoRESUMO
Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme.
Assuntos
Bacillus cereus/enzimologia , Corynebacterium glutamicum/metabolismo , Engenharia Genética/métodos , Fosfolipases Tipo C/biossíntese , Técnicas de Cultura Celular por Lotes , Contagem de Células , Cromatografia Líquida de Alta Pressão , DNA/metabolismo , Fermentação , Expressão Gênica , Vetores Genéticos/metabolismo , Fosfolipases Tipo C/química , Fosfolipases Tipo C/isolamento & purificação , Fosfolipases Tipo C/metabolismoRESUMO
In general, the phospholipase C (PLC) signaling pathway is involved in many physiological activities, including cell growth. However, little is known regarding how the PLC signaling pathway participates in regulating hepatocyte (HC) growth during liver regeneration (LR). To further explore the influence of the PLC signaling pathway on HCs at the cellular level, HCs of high purity and vitality were isolated using Percoll density-gradient centrifugation after partial hepatectomy. The genes of the PLC signaling pathway and target genes of transcription factors in the pathway were obtained by searching the pathways and transcription factor databases, and changes in gene expression of isolated HCs were examined using the Rat Genome 230 2.0 Microarray. The results suggested that various genes involved in the pathway (including 151 known genes and 39 homologous genes) and cell growth (including 262 known genes and 37 homologous genes) were associated with LR. Subsequently, the synergetic effect of these genes in LR was analyzed using a mathematical model (Et) according to their expression profiles. The results showed that the Et values of G protein-coupled receptor/PLC, integrin/PLC, and growth factor receptor/PLC branches of the PLC pathway were all significantly strengthened during the progression and termination phases of LR. The synergetic effect of target genes, in parallel with target gene-related cell growth, was also enhanced during whole rat LR, suggesting the potential positive effect of PLC on HC growth. The present data indicate that the PLC signaling pathway may promote HC growth through 3 mechanisms during rat LR after partial hepatectomy.
Assuntos
Regeneração Hepática/genética , Transdução de Sinais/genética , Fosfolipases Tipo C/genética , Animais , Proliferação de Células/genética , Hepatócitos/metabolismo , Fígado/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Fatores de Transcrição/genética , Fosfolipases Tipo C/isolamento & purificação , Fosfolipases Tipo C/metabolismoRESUMO
Phosphatidylinositol (PtdIns) is a major phospholipid in eukaryotic cells. Many studies have revealed that the phosphoinositide (PI) signaling pathway plays an important role in plant growth and development. Phospholipase C (PLC) is reported to have a crucial role in the PI pathway. This work focuses on the isolation and investigation of PLC in response to abiotic stress factors in green gram. The PLC cDNA, designated VrPLC, encoding a protein of 591 amino acids was cloned and expressed in E. coli. The predicted isoelectric point (pI) and molecular weight were 5.96 and 67.3 kDa, respectively. The tertiary structure of the PLC was also predicted and found to be mainly composed of random coils. In addition, VrPLC expression analysis was performed under environmental stress and the results showed that the expression of VrPLC was rapidly induced in an abscisic acid independent manner in response to drought and salt stress. PLC expression was found to be up-regulated by SA and down-regulated by wound in leaf tissues; however, there was no significant difference in the expression of PLC in plants subjected to high temperature and H2O2. Our results suggest that a close link/relationship between PLC expression and stress responses in green gram.
Assuntos
Fabaceae/enzimologia , Proteínas de Plantas/biossíntese , Estresse Fisiológico/genética , Fosfolipases Tipo C/biossíntese , Sequência de Aminoácidos , Estabilidade Enzimática , Escherichia coli/genética , Fabaceae/genética , Fabaceae/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Fosfolipases Tipo C/química , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/isolamento & purificaçãoRESUMO
Toxicity of the extracellular products (ECPs) and the lethal attributes of phospholipase secreted by pathogenic Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum was studied. An extracellular lethal toxin in the ECPs was partially purified by using Fast Protein Liquid Chromatography system. A protein band (27 kDa) exhibited phospholipase activity on Native-PAGE (by 0.3% egg yolk agar-overlay), was excised and eluted. The pI value of the purified phospholipase was determined as 3.65 and was determined as a phospholipase C by using the Amplex™ Red phosphatidylcholine -Specific phospholipase C Assay kit. The phospholipase showed maximum activity at temperature around 4-40 °C and maximal activity at pH between 8 and 9. The enzyme was inhibited by ethylenediamine-tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS); but was activated by Ca(2+) and Mg(2+) and inactivated by Zn(2+) and Cu(2+) . Both the ECPs and phospholipase were hemolytic against erythrocytes of cobia and lethal to the fish with LD50 values of 3.25 and 0.91 µg protein g(-1) fish, respectively. In toxicity neutralization test, the rabbit antisera against the phospholipase could neutralize the toxicity of ECPs, indicating that the phospholipase is a major extracellular toxin produced by the bacterium.
Assuntos
Peixes/microbiologia , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/metabolismo , Photobacterium/enzimologia , Fosfolipases Tipo C/isolamento & purificação , Fosfolipases Tipo C/metabolismo , Animais , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidade , Hemólise , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Dose Letal Mediana , Peso Molecular , Temperatura , Fosfolipases Tipo C/química , Fosfolipases Tipo C/toxicidadeRESUMO
OBJECTIVE: In this study, we constructed two recombinant Escherichia coli strains to produce phospholipase C (PLC) from Acinetobacter calcoaceticus. The recombinant enzymes were purified to homogeneity and characterized. [Methods] We cloned the PLC encoding gene plc1, plc2 from genome DNA of A. calcoaceticus ATCC17902. The amplified fragments were inserted into pET28a(+ to obtain expression plasmids. E. coli BL21 (DE3) harboring the above plasmids were cultivated and induced with isopropyl-beta-D-thiogalactopyranoside to express PLCs. The recombinant PLCs were purified by affinity chromatography and their catalytic properties were characterized. RESULTS: Two PLCs from A. calcoaceticus were cloned and functional expressed in E. coli. The recombinant enzymes have activities of 31,160 +/- 418 U/mg for PLC1 and 13640 +/- 354 U/mg for PLC2, when using p-nitrophenyl phosphorycholine as substrate. The purified PLC1 and PLC2 exhibited optimum temperature at 65 degrees C and 50 degrees C, respectively. Their optimal pH were 8 and 7.5, respectively. PLC2 was stable under 40 degrees C and pH at 8, whereas the residual activity of PLC1 was less than 25% in the same condition. Mg2+ and Ca2+ stimulated two enzymes activity, whereas Zn2. stimulated PLC1 and inhibited PLC2. PLC1 and PLC2 hydrolyzed phosphatidylinositol. CONCLUSION: It is the first time to express and characterize the PLC gene from A. calcoaceticus ATCC17902. These research results provide reference for the study of food-safety microbiological PLC.
Assuntos
Acinetobacter calcoaceticus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Fosfolipases Tipo C/química , Fosfolipases Tipo C/isolamento & purificação , Acinetobacter calcoaceticus/química , Acinetobacter calcoaceticus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Cinética , Especificidade por Substrato , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17 kDa chaperone (PlcR2) and secreted as a fully folded heterodimer (PlcHR2) of approximately 95 kDa, by the twin arginine translocase (TAT) via the cytoplasmic membrane and through the outer membrane, by the Xcp (TypeII) secretory system. PlcHR2 has been shown to be an important virulence factor in model P. aeruginosa infections and is selectively cytotoxic, at picomolar concentrations to mammalian endothelial cells. Here we report how the various challenges starting from protein overexpression in the native organism P. aeruginosa, the use of detergents in the crystallization and data collection using the most advanced µ-focus synchrotron beam lines were overcome. Native diffraction data of this heterodimeric protein complex were collected up to a resolution of 4Å, whereas needle-shaped crystals of l-selenomethionine substituted PlcHR2 with a maximum diameter of 10 micron were used to collect data sets with a maximum resolution of 2.75Å.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Pseudomonas aeruginosa/enzimologia , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Pseudomonas aeruginosa/metabolismo , Selenometionina/química , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/isolamento & purificação , Esfingomielina Fosfodiesterase/metabolismo , Fosfolipases Tipo C/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/isolamento & purificação , Fatores de Virulência/metabolismoRESUMO
The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Sequência de Bases , Bioensaio , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/isolamento & purificação , Linhagem Celular , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Clostridium perfringens/química , Cães , Eletroforese em Gel de Poliacrilamida , Eritrócitos , Escherichia coli , Vetores Genéticos/metabolismo , Hemólise , Histidina/metabolismo , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/isolamento & purificaçãoRESUMO
Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function.
Assuntos
Membrana Celular/metabolismo , Receptores Nicotínicos/isolamento & purificação , Receptores Nicotínicos/metabolismo , Torpedo/metabolismo , Fosfolipases Tipo C/isolamento & purificação , Fosfolipases Tipo C/metabolismo , Animais , Cromatografia de Afinidade , Cromatografia em Camada Fina , Diglicerídeos/farmacologia , Etilmaleimida/farmacologia , Ácidos Fosfatídicos/metabolismo , Reagentes de Sulfidrila/farmacologiaRESUMO
A novel extracellular phospholipase C (PLC) was purified from a marine streptomycete. It had a molecular mass of 28 kDa as estimated by SDS-polyacrylamide gel electrophoresis. Its enzyme activity was optimal at pH 8.0 at 45 degrees Celsius. The PLC hydrolyzed only phosphatidylcholine. Its activity was enhanced 300% by Na(+) (200 mM), suggesting that the purified PLC is a typical marine-type enzyme.
Assuntos
Espaço Extracelular/enzimologia , Biologia Marinha , Streptomyces/enzimologia , Fosfolipases Tipo C/metabolismo , Cromatografia DEAE-Celulose , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Hidrólise , Fosfolipases Tipo C/isolamento & purificaçãoRESUMO
Only a few methods exist for simple, sensitive and rapid detection of alpha-toxin in clinical and biological samples. The aim of our study was to establish a procedure for the production of an antibody against a recombinant antigen with confirmed sequence identity. We applied a noble approach based on proteomics using a mass spectrometer for the conclusive identification of the recombinant alpha-toxin that was subsequently used as an antigen. The recombinant alpha-toxin was produced in Escherichia coli. A clinical isolate of Clostridium perfringens GAI 94074 was amplified by polymerase chain reaction (PCR) and subsequently, cloning was performed. Three different fragments were cloned using a pET100/D-TOPO vector. These fragments coded for a ribosome binding site, a signal peptide and the alpha-toxin gene, respectively. Recombinant pET100 plasmids were cloned into TOP 10 cells and the isolated plasmids were transferred into BL21 Star (DE3) cells. Their expression was then induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Recombinant E. coli transformed with a plasmid encoding the alpha-toxin gene alone produced a biologically inactive protein. On the other hand, E. coli carrying the plasmid encoding the toxin sequence and its native signal peptide sequence, or the toxin sequence along with the ribosome binding sequence and the signal peptide sequence secreted an active alpha-toxin with phospholipase activity. Accordingly, the C. perfringens gene encoding the alpha-toxin protein along with its signal peptide was successfully cloned, expressed, and secreted by E. coli. Furthermore, without consideration of its activity, we used mass spectrometry to confirm that the expressed protein was indeed the alpha-toxin. Thus, the identification of alpha-toxin protein using both the biological activity testing and the mass spectrometry analysis is expected to verify the significant production of C. perfringens antibody. The study for the analysis of recombinant alpha-toxin using ESI/MS has not been reported. In this study, we report the successful cloning, expression, secretion, identification and sequence determination of the C. perfringens alpha-toxin.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Proteínas de Ligação ao Cálcio/isolamento & purificação , Fosfolipases Tipo C/isolamento & purificação , Toxinas Bacterianas/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Clonagem de Organismos , Clostridium perfringens , Escherichia coli , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Fosfolipases Tipo C/imunologiaRESUMO
Marine bacterial isolates were screened for phospholipase C (PLC) activity on PCY agar plates containing phosphatidylcholine (PC) as substrate. The strain that showed the highest activity on a PCY screening agar plate and a thin-layer chromatography was identified as a strain of Pseudoalteromonas and subsequently designated Pseudoalteromonas sp. J937. The extracellular PLC of the strain J937 was purified to a specific activity of 33 U mg(-1) protein by serial ion exchange and gel filtration column chromatography. It had a molecular mass of 32 kDa estimated by SDS-PAGE. The optimal pH and temperature of the enzyme were about pH 8 and 45 degrees C, respectively. The PLC hydrolyzed phosphatidylethanolamine as well as PC but not other glycerophospholipids. Its activity was enhanced by 150% with Ca2+ (200 mM) and by 180% with Na+ (500 mM), suggesting that the purified PLC is a marine-type enzyme.
Assuntos
Espaço Extracelular/enzimologia , Pseudoalteromonas/enzimologia , Água do Mar/microbiologia , Fosfolipases Tipo C/isolamento & purificação , Estabilidade Enzimática/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Glicerofosfolipídeos/metabolismo , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Metais/farmacologia , Pseudoalteromonas/efeitos dos fármacos , Pseudoalteromonas/isolamento & purificação , Especificidade por Substrato/efeitos dos fármacos , TemperaturaRESUMO
Phospholipase C (PLC) plays an important role in intracellular signal transduction by hydrolyzing phosphatidylinositol 4,5-bis-phosphate, a membrane phospholipid. Currently, thirteen mammalian PLC isozymes have been identified, which are divided into six classes on the basis of structure and mechanisms. All the PLC isozymes share common domains including catalytic X and Y domains, protein kinase C conserved region 2 (C2) domain, EF-hand motif and pleckstrin homology (PH) domain. In this study, the PLC-eta1 PH domain has been over-expressed and purified. The most undesirable feature of the protein was instability, resulting in precipitation during the purification process. With the aim of structural characterization, a solution condition was optimized using SDS-PAGE and NMR spectroscopy. A circular dichroism spectrum indicated that the PLC-eta1 PH domain mainly comprised beta-strands, which was also suggested by the 2D 1H-15N HSQC spectrum.
Assuntos
Proteínas Sanguíneas/química , Fosfoinositídeo Fosfolipase C/química , Fosfoinositídeo Fosfolipase C/metabolismo , Fosfoproteínas/química , Fosfolipases Tipo C/química , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/metabolismo , Dicroísmo Circular , Isoenzimas/química , Isoenzimas/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Dados de Sequência Molecular , Fosfoinositídeo Fosfolipase C/isolamento & purificação , Fosfoproteínas/metabolismo , Estrutura Terciária de Proteína , Fosfolipases Tipo C/isolamento & purificaçãoRESUMO
This study reports the initial separation of phospholipase C-alpha from porcine retina using non-denaturing two-dimensional gel electrophoresis (2-DE). Detection was by negative staining and then its hydrolytic activity was estimated using alpha-naphthyl acetate in a 2-DE gel. A spot of phospholipase C-alpha separated by 2-DE was excised. It was then electrophoretically transferred to an anion-exchange solid phase column after 40 mg, equal to dry weight of the solid resin from the cartridge (Accell Plus QMA, Waters Corporation), was packed into a disposable 1 ml syringe to make an anion-exchange solid phase column. Phosphatidylcholine was hydrolyzed in the anion-exchange solid phase column containing phospholipase C-alpha. The results indicated that a column with hydrolytic activity could be produced once lipases separated by non-denaturing 2-DE were transferred to the solid phase column.
Assuntos
Fosfolipases Tipo C/isolamento & purificação , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia por Troca Iônica , Eletroforese em Gel Bidimensional , Hidrólise , Resinas de Troca Iônica , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fosfatidilcolinas/metabolismo , Retina/enzimologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , Espectrometria de Massas em Tandem , Fosfolipases Tipo C/químicaRESUMO
Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70 degrees C) at acidic pH (pH 3.5-6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g(-1) wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg(-1) protein.
Assuntos
Bacillus cereus/enzimologia , Bacillus subtilis/genética , Fosfolipases Tipo C/biossíntese , Bacillus cereus/genética , Bacillus subtilis/enzimologia , Clonagem Molecular , DNA Bacteriano/genética , Estabilidade Enzimática , Expressão Gênica , Vetores Genéticos , Temperatura Alta , Concentração de Íons de Hidrogênio , Fosforilcolina/análogos & derivados , Fosforilcolina/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Homologia de Sequência de Aminoácidos , Transformação Bacteriana , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/isolamento & purificaçãoRESUMO
This paper uses phospholipase Cepsilon as a model to demonstrate that lipids can act as ligands to bind to specific motifs and regulate protein activity via allosteric effects. Phospholipids such as phosphatidic acid and free fatty acids such as arachidonate are potent activators of PLCepsilon, increasing the rate of PI hydrolysis by 8-fold and 50-fold, respectively. The mechanism appears to be a reduction of K(m), as the substrate dependence curve is shifted to the left and K(m) is reduced 10-fold. The regulation of PLCepsilon by lipids appears to be physiologic, as reconstitution or cotransfection of either cPLA(2) or PLD with PLCepsilon leads to activation of phosphodiesterase activity. Additionally, TSA-201 cells transfected with PLCepsilon and fed arachidonic acid complexed with BSA had increased (4-5-fold) hydrolysis of polyphosphoinositides. This study demonstrates the ability of lipids to act as potent and direct mediators of protein function and identifies cross talk between different classes of phospholipase (PLD and PLA(2) with PLC) mediated via lipid products.
Assuntos
Ácidos Graxos/metabolismo , Fosfolipase D/metabolismo , Fosfolipases A/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Cálcio/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Hidrólise , Ácidos Fosfatídicos/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfoinositídeo Fosfolipase C , Fosfolipase D/genética , Fosfolipases A/genética , Fosfolipases A2 , Fosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Mapeamento de Interação de Proteínas , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/isolamento & purificaçãoRESUMO
Phospholipase C-beta (PLC-beta) isoenzymes are key effectors in G protein-coupled signaling pathways. Prior research suggests that some isoforms of PLC-beta may exist and function as dimers. Using coimmunoprecipitation assays of differentially tagged PLC-beta constructs and size-exclusion chromatography of native PLC-beta, we observed homodimerization of PLC-beta3 and PLC-beta1 isoenzymes but failed to detect heterodimerization of these isoenzymes. Size-exclusion chromatography data suggest that PLC-beta3 and PLC-beta1 form higher affinity homodimers than PLC-beta2. Evidence supportive of limited PLC-beta monomer-homodimer equilibrium appears at < or =100 nM. Further assessment of homodimerization status by coimmunoprecipitation assays with differentially tagged PLC-beta3 fragments demonstrated that at least two subdomains of PLC-beta3 are involved in dimer formation, one in the catalytic X and Y domains and the other in the G protein-regulated carboxyl-terminal domain. In addition, we provide evidence consistent with the existence of PLC-beta homodimers in a whole-cell context, using fluorescent protein-tagged constructs and microscopic fluorescence resonance energy transfer assays.