[Objective] This study aimed to investigate the expression of exogenous gene in prokaryotes and eukaryotes. [Method] The expression vector pTYB2-WF harboring target gene was transformed into E. coli ER2566. IPTG was ...[Objective] This study aimed to investigate the expression of exogenous gene in prokaryotes and eukaryotes. [Method] The expression vector pTYB2-WF harboring target gene was transformed into E. coli ER2566. IPTG was employed to induce the expression of phytase gene. The expression of phytase fusion protein was detected by SDS-PAGE, and the fusion protein was further purified. Phytase gene phyA was expressed in Pichia pastoris expression system. Yeast recombinant vector pPIC9K-phyA was constructed and transformed into P. pastoris GS115 to construct engineering strain GS115-pPIC9K-phyA. [Result] Phytase protein was ex-pressed under methanol induction. Enzyme activity assay indicated that the activity of phytase was 7.3 U/ml. P. pastoris engineering strain GS115-pPIC9K-phyA was successful y constructed. [Conclusion] Methanol yeast expression mechanisms play a certain role in molecular biology and industrial applications.展开更多
The phyA^m gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichiapastoris in order to expand the pH profile o...The phyA^m gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichiapastoris in order to expand the pH profile ofphytase and decrease the cost of production. The fusion phytase phyA^m-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is (25.4±0.53) U/ml at the flask scale and (159.1±2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 ℃ and an optimal pH at 5.5-6.0 and its relative activity remains at a relatively high level of above 70% in the range ofpH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 ℃ to 95 ℃ for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoHf), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those ofphyCs andphyA^m.展开更多
Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase an...Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase and butyrylcholinesterase(BChE, EC 3.1.1.8), which are both candidates for rapid detection of organophosphate pesticides. To develop an easy-tohandle method for detecting organophosphate pesticides using BChE, BChE from human was optimized according to the codon usage bias of Pichia pastoris and successfully expressed in P. pastoris GS115. The codon-optimized cDNA shared 37.3% of the codon identity with the native one. However, the amino acid sequence was identical to that of the native human butyrylcholinesterase gene(h BCh E) as published. The ratio of guanine and cytosine in four kinds of bases((G+C) ratio) was simultaneously increased from 40 to 47%. The recombinant hBChE expression reached a total protein concentration of 292 mg m L^–1 with an activity of 14.7 U m L^–1, which was purified 3.2×10^3-fold via nickel affinity chromatography with a yield of 68% and a specific activity of 8.1 U mg^–1. Recombinant hBChE was optimally active at pH 7.4 and 50°C and exhibited high activity at a wide pH range(〉60% activity at pH 4.0 to 8.0). Moreover, it had a good adaptability to high temperature(〉60% activity at both 50 and 60°C up to 60 min) and good stability at 70°C. The enzyme can be activated by Li^+, Co^+, Zn^2+ and ethylene diamine tetraacetic acid(EDTA), but inhibited by Mg^2+, Mn^2+, Fe^2+, Ag^+ and Ca^2+. Na^+ had little effect on its activity. The values of h BChE of the Michaelis constant(Km) and maximum reaction velocity(Vm) were 89.4 mmol L^–1 and 1 721 mmol min^–1 mg^–1, respectively. The bim olecular rate constants(K_i) of the hBChE to four pesticides were similar with that of electric eel AChE(EeAChE) and higher than that of horse BChE(HoBChE). All vlues of the half maximal inhibitory concentration of a substance(IC50) for hBChE were lower than those for HoBChE, but most IC50 for hBChE were lower than those for EeAChE except dichlorvos. The applicability of the hBChE was further verified by successful detection of organophosphate insecticide residues in six kinds of vegetable samples. Thus, hBChE heterologously over-expressed by P. pastoris would provide a sufficient material for development of a rapid detection method of organophosphate on spot and produce the organophosphate detection kit.展开更多
The mannose-binding lectin GNA (snowdrop lectin) is used as a"carrier" domain in insecticidal fusion proteins which cross the insect gut after oral ingestion. A similar lectin from garlic bulb, ASAII, has been eva...The mannose-binding lectin GNA (snowdrop lectin) is used as a"carrier" domain in insecticidal fusion proteins which cross the insect gut after oral ingestion. A similar lectin from garlic bulb, ASAII, has been evaluated as an alternative "carrier". Recombinant ASAII delivered orally to larvae of cabbage moth (Mamestra brassica; Lepidoptera) was subsequently detected in haemolymph, demonstrating transport. Fusion proteins comprising an insect neurotoxin, ButaIT (Buthus tamulus insecticidal toxin; red scorpion toxin) linked to the C-terminal region of ASAII or GNA were produced as recombinant proteins (GNA/ ButaIT and ASA/ButaIT) by expression in Pichia pastoris. In both cases the C-terminal sequence of the lectin was truncated to avoid post-translational proteolysis. The GNA- containing fusion protein was toxic by injection to cabbage moth larvae (LD50≈ 250μg/g), and when fed had a negative effect on survival and growth. It also decreased the survival of cereal aphids (Sitobion avenae; Homoptera) from neonate to adult by 〉70% when fed. In contrast, the ASA-ButaIT fusion protein was non-toxic to aphids, and had no effect on lepidopteran larvae, either when injected or when fed. However, intact ASA-ButaIT fusion protein was present in the haemolymph of cabbage moth larvae following ingestion, showing that transport of the fusion had occurred. The stabilities of GNA/ButaIT and ASA/ButaIT to proteolysis in vivo after injection or ingestion differed, and this may be a factor in determining insecticidal activities.展开更多
文摘[Objective] This study aimed to investigate the expression of exogenous gene in prokaryotes and eukaryotes. [Method] The expression vector pTYB2-WF harboring target gene was transformed into E. coli ER2566. IPTG was employed to induce the expression of phytase gene. The expression of phytase fusion protein was detected by SDS-PAGE, and the fusion protein was further purified. Phytase gene phyA was expressed in Pichia pastoris expression system. Yeast recombinant vector pPIC9K-phyA was constructed and transformed into P. pastoris GS115 to construct engineering strain GS115-pPIC9K-phyA. [Result] Phytase protein was ex-pressed under methanol induction. Enzyme activity assay indicated that the activity of phytase was 7.3 U/ml. P. pastoris engineering strain GS115-pPIC9K-phyA was successful y constructed. [Conclusion] Methanol yeast expression mechanisms play a certain role in molecular biology and industrial applications.
基金the National Key Technologies R & D Program of China during the 10th Five-Year Plan Period (No. 2002BA514A-12)the Education Department of Sichuan Province (No. 2006B014)the Innovative Fund for Distinguished Young Scholars of Sichuan Agricultural University, China
文摘The phyA^m gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichiapastoris in order to expand the pH profile ofphytase and decrease the cost of production. The fusion phytase phyA^m-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is (25.4±0.53) U/ml at the flask scale and (159.1±2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 ℃ and an optimal pH at 5.5-6.0 and its relative activity remains at a relatively high level of above 70% in the range ofpH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 ℃ to 95 ℃ for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoHf), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those ofphyCs andphyA^m.
基金financially supported by the Rural Work Committee of Beijing City for the First Batch of Agricultural Science and Technology Project of Beijing University of Agriculture, China (2013010102)the Beijing Innovation Team Building Project of Leafy Vegetables of Modern Agricultural Industry System, China (2063213003)the Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions, China (CIT&TCD 20150315)
文摘Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase and butyrylcholinesterase(BChE, EC 3.1.1.8), which are both candidates for rapid detection of organophosphate pesticides. To develop an easy-tohandle method for detecting organophosphate pesticides using BChE, BChE from human was optimized according to the codon usage bias of Pichia pastoris and successfully expressed in P. pastoris GS115. The codon-optimized cDNA shared 37.3% of the codon identity with the native one. However, the amino acid sequence was identical to that of the native human butyrylcholinesterase gene(h BCh E) as published. The ratio of guanine and cytosine in four kinds of bases((G+C) ratio) was simultaneously increased from 40 to 47%. The recombinant hBChE expression reached a total protein concentration of 292 mg m L^–1 with an activity of 14.7 U m L^–1, which was purified 3.2×10^3-fold via nickel affinity chromatography with a yield of 68% and a specific activity of 8.1 U mg^–1. Recombinant hBChE was optimally active at pH 7.4 and 50°C and exhibited high activity at a wide pH range(〉60% activity at pH 4.0 to 8.0). Moreover, it had a good adaptability to high temperature(〉60% activity at both 50 and 60°C up to 60 min) and good stability at 70°C. The enzyme can be activated by Li^+, Co^+, Zn^2+ and ethylene diamine tetraacetic acid(EDTA), but inhibited by Mg^2+, Mn^2+, Fe^2+, Ag^+ and Ca^2+. Na^+ had little effect on its activity. The values of h BChE of the Michaelis constant(Km) and maximum reaction velocity(Vm) were 89.4 mmol L^–1 and 1 721 mmol min^–1 mg^–1, respectively. The bim olecular rate constants(K_i) of the hBChE to four pesticides were similar with that of electric eel AChE(EeAChE) and higher than that of horse BChE(HoBChE). All vlues of the half maximal inhibitory concentration of a substance(IC50) for hBChE were lower than those for HoBChE, but most IC50 for hBChE were lower than those for EeAChE except dichlorvos. The applicability of the hBChE was further verified by successful detection of organophosphate insecticide residues in six kinds of vegetable samples. Thus, hBChE heterologously over-expressed by P. pastoris would provide a sufficient material for development of a rapid detection method of organophosphate on spot and produce the organophosphate detection kit.
文摘The mannose-binding lectin GNA (snowdrop lectin) is used as a"carrier" domain in insecticidal fusion proteins which cross the insect gut after oral ingestion. A similar lectin from garlic bulb, ASAII, has been evaluated as an alternative "carrier". Recombinant ASAII delivered orally to larvae of cabbage moth (Mamestra brassica; Lepidoptera) was subsequently detected in haemolymph, demonstrating transport. Fusion proteins comprising an insect neurotoxin, ButaIT (Buthus tamulus insecticidal toxin; red scorpion toxin) linked to the C-terminal region of ASAII or GNA were produced as recombinant proteins (GNA/ ButaIT and ASA/ButaIT) by expression in Pichia pastoris. In both cases the C-terminal sequence of the lectin was truncated to avoid post-translational proteolysis. The GNA- containing fusion protein was toxic by injection to cabbage moth larvae (LD50≈ 250μg/g), and when fed had a negative effect on survival and growth. It also decreased the survival of cereal aphids (Sitobion avenae; Homoptera) from neonate to adult by 〉70% when fed. In contrast, the ASA-ButaIT fusion protein was non-toxic to aphids, and had no effect on lepidopteran larvae, either when injected or when fed. However, intact ASA-ButaIT fusion protein was present in the haemolymph of cabbage moth larvae following ingestion, showing that transport of the fusion had occurred. The stabilities of GNA/ButaIT and ASA/ButaIT to proteolysis in vivo after injection or ingestion differed, and this may be a factor in determining insecticidal activities.