Pectin lyase(PMGL)is an industrially important enzyme with widespread applications in the food,paper,and textile industries,owing to its capacity for direct degradation of highly esterified pectin.In this study,PMGL-B...Pectin lyase(PMGL)is an industrially important enzyme with widespread applications in the food,paper,and textile industries,owing to its capacity for direct degradation of highly esterified pectin.In this study,PMGL-Ba derived from Bacillus licheniformis underwent mining and heterologous expression in P.pastoris.Furthermore,diverse strategies,encompassing the optimization of expression cassette components,elevation of gene dosage,and co-expression of chaperone factors,were employed to augment PMGL-Ba production in P.pastoris.The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements.By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup,a strain yielding high PMGL-Ba production was achieved.In shake flask fermentation lasting 144 h,the total protein concentration reached 1.81 g/L,and the enzyme activity reached 1821.36 U/mL.For further scale up production,high-density fermentation transpired in a 5 L fermenter for 72 h.Remarkably,the total protein concentration increased to 12.49 g/L,and the enzyme activity reached an impressive 12668.12 U/mL.The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications.展开更多
To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the con...To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the control of promoters： RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae： RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ. In addition, a threefold increase of GFP in Tran-Ⅱ was induced by heat shock at 40℃. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.展开更多
Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affe...Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.展开更多
The number of therapeutic monoclonal antibodies used in clinical trials has recently increased dramatically, leading to the development of optimized downstream purification processes[1]. Staphylococcal protein A （SPA...The number of therapeutic monoclonal antibodies used in clinical trials has recently increased dramatically, leading to the development of optimized downstream purification processes[1]. Staphylococcal protein A （SPA）, a cell-wall protein of Staphylococcus aureus, has been developed as a universal ligand for immunoglobulin G （IgG） purification because it binds specifically to the Fc portion of the IgG molecule of many mammals[2]. However, certain characteristics of SPA severely restrict the advancement of the antibody industry.展开更多
Bit Error Probability (BEP) provides a fundamental performance measure for wireless diversity systems. This paper presents two new exact BEP expressions for Maximal Ratio Combining (MRC) diversity systems. One BEP exp...Bit Error Probability (BEP) provides a fundamental performance measure for wireless diversity systems. This paper presents two new exact BEP expressions for Maximal Ratio Combining (MRC) diversity systems. One BEP expression takes a closed form, while the other is derived by treating the squared-sum of Rayleigh random variables as an Erlang variable. Due to the fact that the extant bounds are loose and could not properly characterize the error performance of MRC diversity systems, this paper presents a very tight bound. The numerical analysis shows that the new derived BEP expressions coincide with the extant expressions, and that the new approximation tightly bounds the accurate BEP.展开更多
Human erythropoietin gene was ligated into the mammalian high efficiency expression vector pSV2 dhfr by standard procedures of filling in, trimming, ligation and transformation to construct a high efficiency expressi...Human erythropoietin gene was ligated into the mammalian high efficiency expression vector pSV2 dhfr by standard procedures of filling in, trimming, ligation and transformation to construct a high efficiency expression vector. After the expression vector pSEPO25 was transfected into CHO dhfr - cells, the cell line which could express high levels of EPO was successfully selected. The result lays the foundation for production of EPO by genetic biotechnology.展开更多
Functional saccharide is a general term that is often used to refer to the functional oligosaccharides,functional saccharide alcohols,and functional dietary fibers.These functional saccharides exhibit some health bene...Functional saccharide is a general term that is often used to refer to the functional oligosaccharides,functional saccharide alcohols,and functional dietary fibers.These functional saccharides exhibit some health benefiting effects,such as having low calorie,preventing dental caries,and regulating intestinal disorders.Functional saccharides are widely used in food,health products,and the healthcare fields.The preparation of functional saccharides is accomplished mainly through reactions involving transglycosylation,isomerization,or hydrolysis catalyzed by glycosyltransferases,saccharide isomerases,and glycohydrolases,respectively.However,the poor catalytic properties of natural enzymes and low fermentation yields have restricted the large-scale industrial production of functional saccharides.Therefore,molecular modification and efficient expression of key enzymes for functional-saccharide preparation are very important for promoting the low-cost large-scale production of functional saccharides.In this report,the recent advances in functional optimization and expression preparation of enzymes related to functional saccharides are reviewed.展开更多
基金supported by the National Key R&D Program of China(NO.2021YFC2100400).
文摘Pectin lyase(PMGL)is an industrially important enzyme with widespread applications in the food,paper,and textile industries,owing to its capacity for direct degradation of highly esterified pectin.In this study,PMGL-Ba derived from Bacillus licheniformis underwent mining and heterologous expression in P.pastoris.Furthermore,diverse strategies,encompassing the optimization of expression cassette components,elevation of gene dosage,and co-expression of chaperone factors,were employed to augment PMGL-Ba production in P.pastoris.The signaling peptide OST1-pre-α-MF-pro and promoter AOX1 were finally selected as expression elements.By overexpressing the transcription factor Hac1p in conjunction with a two-copy PMGL-Ba setup,a strain yielding high PMGL-Ba production was achieved.In shake flask fermentation lasting 144 h,the total protein concentration reached 1.81 g/L,and the enzyme activity reached 1821.36 U/mL.For further scale up production,high-density fermentation transpired in a 5 L fermenter for 72 h.Remarkably,the total protein concentration increased to 12.49 g/L,and the enzyme activity reached an impressive 12668.12 U/mL.The successful heterologous and efficient expression of PMGL-Ba not only furnishes a valuable biological enzyme for industrial applications but also contributes to cost reduction in the utilization of biological enzymes in industrial applications.
基金Supported by the High Technology Research and Development Program of China (863 Program) (No. 2005AA601010-05)the Natural Science Foundation of Guangdong Province (No.5010492)the Technology Project of Shenzhen City
文摘To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the control of promoters： RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae： RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ. In addition, a threefold increase of GFP in Tran-Ⅱ was induced by heat shock at 40℃. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.
基金financed by the National Natural Science Foundation of China (30900972, 31572111)the Special Found for Agro-scientific Research in the Public Interest, China (201203076-06)the Graduate Innovative Projects of Hunan Province, China (CX2013B290)
文摘Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.
基金supported by grants from the National High Technology Research and Development Program of China(863 Program)(No.2012AA020304)the National Natural Science Foundation of China(Grant No.31300643)the Innovation Fund for Postgraduate Students from the Simcere Pharmaceutical Company(No.02704051)
文摘The number of therapeutic monoclonal antibodies used in clinical trials has recently increased dramatically, leading to the development of optimized downstream purification processes[1]. Staphylococcal protein A （SPA）, a cell-wall protein of Staphylococcus aureus, has been developed as a universal ligand for immunoglobulin G （IgG） purification because it binds specifically to the Fc portion of the IgG molecule of many mammals[2]. However, certain characteristics of SPA severely restrict the advancement of the antibody industry.
基金Supported by the National Natural Science Foundation of China (No.60572059)Foundation of Guangdong Province for Ph.D. (No. 5300707).
文摘Bit Error Probability (BEP) provides a fundamental performance measure for wireless diversity systems. This paper presents two new exact BEP expressions for Maximal Ratio Combining (MRC) diversity systems. One BEP expression takes a closed form, while the other is derived by treating the squared-sum of Rayleigh random variables as an Erlang variable. Due to the fact that the extant bounds are loose and could not properly characterize the error performance of MRC diversity systems, this paper presents a very tight bound. The numerical analysis shows that the new derived BEP expressions coincide with the extant expressions, and that the new approximation tightly bounds the accurate BEP.
文摘Human erythropoietin gene was ligated into the mammalian high efficiency expression vector pSV2 dhfr by standard procedures of filling in, trimming, ligation and transformation to construct a high efficiency expression vector. After the expression vector pSEPO25 was transfected into CHO dhfr - cells, the cell line which could express high levels of EPO was successfully selected. The result lays the foundation for production of EPO by genetic biotechnology.
文摘Functional saccharide is a general term that is often used to refer to the functional oligosaccharides,functional saccharide alcohols,and functional dietary fibers.These functional saccharides exhibit some health benefiting effects,such as having low calorie,preventing dental caries,and regulating intestinal disorders.Functional saccharides are widely used in food,health products,and the healthcare fields.The preparation of functional saccharides is accomplished mainly through reactions involving transglycosylation,isomerization,or hydrolysis catalyzed by glycosyltransferases,saccharide isomerases,and glycohydrolases,respectively.However,the poor catalytic properties of natural enzymes and low fermentation yields have restricted the large-scale industrial production of functional saccharides.Therefore,molecular modification and efficient expression of key enzymes for functional-saccharide preparation are very important for promoting the low-cost large-scale production of functional saccharides.In this report,the recent advances in functional optimization and expression preparation of enzymes related to functional saccharides are reviewed.
