A genetic recombinant hEGF producer E.coli JM101 was immobilized in polyurethane foam matrix and a three-phase fluidized-bed bioreactor was used to produce human epidermal growth factor (hEGF).The results indicated th...A genetic recombinant hEGF producer E.coli JM101 was immobilized in polyurethane foam matrix and a three-phase fluidized-bed bioreactor was used to produce human epidermal growth factor (hEGF).The results indicated that polyurethane foam is an adequate immobilization material. With the addition of polyurethane foam matrix, the free cell density, plasmid stability and hEGF productivity were higher than those without the foam matrix, respectively. The two-stage culture of the immobilized cells was further carried out, in which, the first stage was performed in a three-phase fluidized-bed bioreactor for cell growth and the second stage was performed in bubbling bed for product expression. At a dilution rate of 0.2 h -1 , after continuous cultivation for more than 60 h, the free cell density and product expression were maintained relatively stable. The hEGF productivity was still higher than 140 mg·L -1 and the fraction of plasmid-carrying was more than 27%. The hEGF productivity in two-stage process was higher than the batch process.展开更多
The effects of different glucose feeding modes on hEGF expression were evaluated in an excretory recombinant \%E.coli\% K12 system.The results showed that,compared with batch cultivation,the plasmid stability and dens...The effects of different glucose feeding modes on hEGF expression were evaluated in an excretory recombinant \%E.coli\% K12 system.The results showed that,compared with batch cultivation,the plasmid stability and density of plasmid\|retaining cells were improved by all three glucose feeding modes (intermittent,pH\|stat and constant\|rate).It was shown that hEGF yields were improved up to 25.5% and 28.1% by intermittent or pH\|stat glucose feeding respectively.Especially,up to 150% improvement of hEGF production was achieved by constant feeding of 200g/L glucose solution at a rate of 0.11 mL/min.The effects of further combined feeding with other medium components (ampicillin,nitrogen sources,and inorganic salts) and inducer on hEGF yield were also examined in the bench\|top fermentor.展开更多
Production and accumulation of toxic by-products such as acetic acid can inhibit the growth of recombinant cells and the expression of exogenous gene in E.coli. An anionic exchange resin, A-D 3-1, which is high in ads...Production and accumulation of toxic by-products such as acetic acid can inhibit the growth of recombinant cells and the expression of exogenous gene in E.coli. An anionic exchange resin, A-D 3-1, which is high in adsorption selectivity and capability for acetic acid, was screened from a variety of resins based on its physical and chemical properties.On the scale of shake flask culture, the addition of 1.0 g resin per 30 ml medium was insignificant for the cell growth, however,it could improve the hEGF expression significantly. The batch culture in 2.5 L fermentor showed that in-situ adsorption of acetic acid by anionic exchange resin could enhance the expression level of interested protein and reduce the fermentation period by 2 hours. And up to 10% improvement of hEGF (human epidermal growth factor)volumetric productivity (225.0 mg·L -1 ) could be achieved by supplementing 3.3 g resin per 100 ml medium.展开更多
ObjectiveThis study aimed to construct plant expression vector for recombinant human epidermal growth factor （hEGF） and further to provide a basis for the expression of hEGF in peanut hairy root system. MethodAccord...ObjectiveThis study aimed to construct plant expression vector for recombinant human epidermal growth factor （hEGF） and further to provide a basis for the expression of hEGF in peanut hairy root system. MethodAccording to the hEGF sequence in GenBank, hEGF was synthesized artificially; subsequently, hEGF gene was ligated with green fluorescent protein （GFP） gene, and their ligation product was then amplified with primers flanked with corresponding endonuclease cleavage sites, followed by double digestion by Sal I and EcoR I of the amplified products; next, pRI 101 AN DNA was extracted and digested by both Sal I and EcoR I; susequently, the digestion products of hEGF and GFP ligation fragment by Sal I and EcoR I and the digestion products of pRI 101 AN plasmid DNA by Sal I and EcoR I were ligated, and their ligation product was transformed into Escherichia coli XL10-Gold, followed by extraction of DNA from the recombinants exhibiting green fluorescence, which was then identified by enzymatic digestion and PCR, and the verified recombinant plasmid DNA was named pBZG101. ResultHuman epidermal growth factor gene （hEGF） and green fluorescent protein gene （GFP） were successfully ligated, and their ligation fragment was successfully ligated to pRI 101 AN DNA, finally with the acquirement of the plant expression vector for recombinant human epidermal growth factor-（pBZG101）. ConclusionThe plant expression vector for recombinant human epidermal growth factor-（pBZG101）- was successfully constructed in this study.展开更多
文摘A genetic recombinant hEGF producer E.coli JM101 was immobilized in polyurethane foam matrix and a three-phase fluidized-bed bioreactor was used to produce human epidermal growth factor (hEGF).The results indicated that polyurethane foam is an adequate immobilization material. With the addition of polyurethane foam matrix, the free cell density, plasmid stability and hEGF productivity were higher than those without the foam matrix, respectively. The two-stage culture of the immobilized cells was further carried out, in which, the first stage was performed in a three-phase fluidized-bed bioreactor for cell growth and the second stage was performed in bubbling bed for product expression. At a dilution rate of 0.2 h -1 , after continuous cultivation for more than 60 h, the free cell density and product expression were maintained relatively stable. The hEGF productivity was still higher than 140 mg·L -1 and the fraction of plasmid-carrying was more than 27%. The hEGF productivity in two-stage process was higher than the batch process.
文摘The effects of different glucose feeding modes on hEGF expression were evaluated in an excretory recombinant \%E.coli\% K12 system.The results showed that,compared with batch cultivation,the plasmid stability and density of plasmid\|retaining cells were improved by all three glucose feeding modes (intermittent,pH\|stat and constant\|rate).It was shown that hEGF yields were improved up to 25.5% and 28.1% by intermittent or pH\|stat glucose feeding respectively.Especially,up to 150% improvement of hEGF production was achieved by constant feeding of 200g/L glucose solution at a rate of 0.11 mL/min.The effects of further combined feeding with other medium components (ampicillin,nitrogen sources,and inorganic salts) and inducer on hEGF yield were also examined in the bench\|top fermentor.
文摘Production and accumulation of toxic by-products such as acetic acid can inhibit the growth of recombinant cells and the expression of exogenous gene in E.coli. An anionic exchange resin, A-D 3-1, which is high in adsorption selectivity and capability for acetic acid, was screened from a variety of resins based on its physical and chemical properties.On the scale of shake flask culture, the addition of 1.0 g resin per 30 ml medium was insignificant for the cell growth, however,it could improve the hEGF expression significantly. The batch culture in 2.5 L fermentor showed that in-situ adsorption of acetic acid by anionic exchange resin could enhance the expression level of interested protein and reduce the fermentation period by 2 hours. And up to 10% improvement of hEGF (human epidermal growth factor)volumetric productivity (225.0 mg·L -1 ) could be achieved by supplementing 3.3 g resin per 100 ml medium.
基金Supported by the Shangdong Natural Science Foundation(ZR2010HQ054)the Ministry of Agriculture Opening Project Fund of Key Laboratory of Rubber Biology/State Key Laboratory Breeding Base of Cultivation&Physiology for Tropical Crops(KLOF1106)the Special Fund for Backbone Teachers and Domestic Visiting Scholars of Shandong Higher Education Institutions9~~
文摘ObjectiveThis study aimed to construct plant expression vector for recombinant human epidermal growth factor （hEGF） and further to provide a basis for the expression of hEGF in peanut hairy root system. MethodAccording to the hEGF sequence in GenBank, hEGF was synthesized artificially; subsequently, hEGF gene was ligated with green fluorescent protein （GFP） gene, and their ligation product was then amplified with primers flanked with corresponding endonuclease cleavage sites, followed by double digestion by Sal I and EcoR I of the amplified products; next, pRI 101 AN DNA was extracted and digested by both Sal I and EcoR I; susequently, the digestion products of hEGF and GFP ligation fragment by Sal I and EcoR I and the digestion products of pRI 101 AN plasmid DNA by Sal I and EcoR I were ligated, and their ligation product was transformed into Escherichia coli XL10-Gold, followed by extraction of DNA from the recombinants exhibiting green fluorescence, which was then identified by enzymatic digestion and PCR, and the verified recombinant plasmid DNA was named pBZG101. ResultHuman epidermal growth factor gene （hEGF） and green fluorescent protein gene （GFP） were successfully ligated, and their ligation fragment was successfully ligated to pRI 101 AN DNA, finally with the acquirement of the plant expression vector for recombinant human epidermal growth factor-（pBZG101）. ConclusionThe plant expression vector for recombinant human epidermal growth factor-（pBZG101）- was successfully constructed in this study.
