The antifungal peptides, drosomycin （Drs） and its isoform drosomycin-like C （Drs-lC） from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamento...The antifungal peptides, drosomycin （Drs） and its isoform drosomycin-like C （Drs-lC） from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-lC, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-lC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-lC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains.展开更多
Antimicrobial peptides （AMPs） are making headlines in science because they demonstrate superior microbicidal characteristics compared to synthetic and semi-synthetic antibiotics.
[ Objective] This study aimed to optimize the cedon of antimicrobial peptide SMAP-29 gene and express SMAP-29 in Pichia pastoris. [ Method] Ac- cording to the published amino acid sequence of SMAP-29, a gene encoding ...[ Objective] This study aimed to optimize the cedon of antimicrobial peptide SMAP-29 gene and express SMAP-29 in Pichia pastoris. [ Method] Ac- cording to the published amino acid sequence of SMAP-29, a gene encoding SMAP-29 （sheep myeloid antibacterial peptides-29） mature peptide was synthesized and was biased codon usage of Pichia pastoris. The synthesized gene was inserted into Pichia pastoris expression vector pPIC3.5K, transformed into Pichia pastoris strain GSll5 by electmporation, and screened with G418 for high-copy recombinants. The methanol utility plus phenotype （Mut ＋ ） was selected with MM, MD and PCR. The correctly constructed recombinant strain was induced with methanol. Expression of SMAP-29 was analyzed by lysing the induced yeast cells with Tricine- SDS-PAGE. [ Result] After induced with methanol for 2 d, a 3.2 kD induced band was detected in the cell lysate which was consistent to the predicted molecular weight of SMAP-29 ; the expression products purified with gel filtration chromatography showed significant antibacterial effect against Staphyloccocus aureus and Mo- nilia albican but no significant antibacterial effect against Escherichia coli. [ Conclusion] This study laid the foundation for the application of SMAP-29 in biomedi- cine, agriculture and other fields.展开更多
The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into ...The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.展开更多
[ Objective ] This study aimed to obtain the recombinant Pichia yeast strain which can efficiently degrade guar gum. The properties of the recombinant enzyme were studied preliminarily. [ Method ] A positive clone tha...[ Objective ] This study aimed to obtain the recombinant Pichia yeast strain which can efficiently degrade guar gum. The properties of the recombinant enzyme were studied preliminarily. [ Method ] A positive clone that could hydrolyze guar gum was obtained through the construction and functional screening of a soil genome library. Sequence analysis indicated that the 1485-bp clone encodes a 494-amino acid protein with a relative molecular mass of 53 949 kD, containing a cellulose-binding domain. The recombinant plasmid pHBM731 was generated by inserting the optimized target gene into a Pichia pastoris expression vector pHBMg05 that was transformed into three Pichia pastoris strains, GS115, KM71 and SMD1168. The biochemical properties of the enzyme were assessed. [ Result] The cloned galactonumnan （GM）-degrading enzyme was expressed and secreted by Pichia pastoris GSll5. High cell density fermentation was induced in recombi- nant Pichia pastoris at 25 and 28 ~C ; a higher enzyme activity was observed at an induction temperature of 28 ~C. The optimal temperature for the recombinant en- zyme is 60 ~C, and the optimal pH is 6.6. The enzyme activity was 38.61 U under optimal conditions. Over 50% of the enzyme activity was maintained under the optimal conditions after 9 h. Under the optimal conditions, the effect of metal ions on enzyme activity was analyzed. Ca2 ＋ , Fe2 ＋ and Li ~ slightly enhanced enzyme activity, while Mn2＋ and Co2＋ had little effect. Enzyme activity was modestly suppressed by Mg2~ , K~ and Na＋ , but considerably suppressed by Ag2~ and Zn2~ , with Cu2 ＋ showing the strongest inhibitory effects. [ Conclusion] A novel GM-degrading enzyme expressed by soil yeast was cloned, which can potentially be used in industrial applications to obtain eommereially useful guar gum-degradation products.展开更多
To obtain human tissue inhibitor of metalloproteinase-2(TIMP-2)cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris,we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 wit...To obtain human tissue inhibitor of metalloproteinase-2(TIMP-2)cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris,we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique,which was composed of frequently used codons in the highly expressed Pichia pastoris genes.Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing.The verified gene of TIMP2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2.The plasmid was transformed into GS115 cells of the methylotrophic yeast,Pichia pastoris by electroporation,and we got the expression cell through phenotype selection and induction with methanol.Separation,purification,and bioactivity analysis of the expressed products were performed.展开更多
T4 lysozyme was engineered with disulfide bonds and expressed in Pichia pastoris. The secreted proteins were purified and made into powder by lyophilization. Recombinant protein purity was more than 70% measured by HP...T4 lysozyme was engineered with disulfide bonds and expressed in Pichia pastoris. The secreted proteins were purified and made into powder by lyophilization. Recombinant protein purity was more than 70% measured by HPLC. The lytic activity of variant T4-lysozyme was measured by the lysis of the cell wall of Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Ralstonia solanacearum comb.nov, Clavibacter michiganensis subsp. michiganensis, X. campestris pv. malvacearum, Fusarium oxysporium sp. vasinfectum, Verticillium dahliae kleb. Inhibition zone assay showed that variant T4 lysozyme significantly inhibited X. o. oryzicola and X. c. malvacearum. The antifungal activities of this protein against F. o. vasinfectum and V. d. kleb were also analyzed.展开更多
To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinan...To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and expression.The biochemical and enzymatic properties of the new recombinant were characterized.As a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,respectively.Under the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation.To further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L bioreactors.Using high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L.In addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(min.mg),respectively.Thus,TrCel5A was effectively expressed in P.pastoris under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.展开更多
利用含有强启动子 PAOX1 和 α-因子信号肽序列的巴斯德毕赤酵母载体 p PICZαA,构建出含牛白细胞介素 2(Bo IL - 2 )基因的重组质粒 Bo IL 2 - p PICZαA。线性化的重组表达载体转化到巴斯德毕赤酵母 X- 33及 KM71H中 ,筛选Zeocin高抗...利用含有强启动子 PAOX1 和 α-因子信号肽序列的巴斯德毕赤酵母载体 p PICZαA,构建出含牛白细胞介素 2(Bo IL - 2 )基因的重组质粒 Bo IL 2 - p PICZαA。线性化的重组表达载体转化到巴斯德毕赤酵母 X- 33及 KM71H中 ,筛选Zeocin高抗性酵母菌株 ,甲醇诱导目的蛋白表达。经 SDS- PAGE和 Western blot检测表明 ,Bo IL- 2以融合蛋白形式在胞内表达 ,但没能分泌到胞外。通过 Bo IL- 2在巴斯德毕赤酵母中的表达 ,重点讨论了信号肽。展开更多
基金the grants from the National Basic Research Program of Ministry of Science and Technology, China （973 Program, 2005CB 121000） the Science and Technology Project of Guangdong Province, China （2003C104042） the Natural Science Foundation of Guangdong Province, China （032256, 04020553）.
文摘The antifungal peptides, drosomycin （Drs） and its isoform drosomycin-like C （Drs-lC） from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-lC, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-lC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-lC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains.
基金supported by grants from the Project of Jilin province Technology Platform(NO.20070210)the Young Research Fond of Jilin University
文摘Antimicrobial peptides （AMPs） are making headlines in science because they demonstrate superior microbicidal characteristics compared to synthetic and semi-synthetic antibiotics.
基金Supported by Basic Research Project from Science and Technology Bureau of Xinjiang Production and Construction Corps (2006JC01)National Natural Science Foundation of China (31160218)
文摘[ Objective] This study aimed to optimize the cedon of antimicrobial peptide SMAP-29 gene and express SMAP-29 in Pichia pastoris. [ Method] Ac- cording to the published amino acid sequence of SMAP-29, a gene encoding SMAP-29 （sheep myeloid antibacterial peptides-29） mature peptide was synthesized and was biased codon usage of Pichia pastoris. The synthesized gene was inserted into Pichia pastoris expression vector pPIC3.5K, transformed into Pichia pastoris strain GSll5 by electmporation, and screened with G418 for high-copy recombinants. The methanol utility plus phenotype （Mut ＋ ） was selected with MM, MD and PCR. The correctly constructed recombinant strain was induced with methanol. Expression of SMAP-29 was analyzed by lysing the induced yeast cells with Tricine- SDS-PAGE. [ Result] After induced with methanol for 2 d, a 3.2 kD induced band was detected in the cell lysate which was consistent to the predicted molecular weight of SMAP-29 ; the expression products purified with gel filtration chromatography showed significant antibacterial effect against Staphyloccocus aureus and Mo- nilia albican but no significant antibacterial effect against Escherichia coli. [ Conclusion] This study laid the foundation for the application of SMAP-29 in biomedi- cine, agriculture and other fields.
基金Supported by National High-tech Research and Development Program of China(No.2007AA021004)
文摘The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.
基金Supported by Yantai Municipal Science and Technology Development Plan(2013ZH097)Scientific and Technological Innovation Fund for Students in Binzhou Medical University(BY2013DKCX122)
文摘[ Objective ] This study aimed to obtain the recombinant Pichia yeast strain which can efficiently degrade guar gum. The properties of the recombinant enzyme were studied preliminarily. [ Method ] A positive clone that could hydrolyze guar gum was obtained through the construction and functional screening of a soil genome library. Sequence analysis indicated that the 1485-bp clone encodes a 494-amino acid protein with a relative molecular mass of 53 949 kD, containing a cellulose-binding domain. The recombinant plasmid pHBM731 was generated by inserting the optimized target gene into a Pichia pastoris expression vector pHBMg05 that was transformed into three Pichia pastoris strains, GS115, KM71 and SMD1168. The biochemical properties of the enzyme were assessed. [ Result] The cloned galactonumnan （GM）-degrading enzyme was expressed and secreted by Pichia pastoris GSll5. High cell density fermentation was induced in recombi- nant Pichia pastoris at 25 and 28 ~C ; a higher enzyme activity was observed at an induction temperature of 28 ~C. The optimal temperature for the recombinant en- zyme is 60 ~C, and the optimal pH is 6.6. The enzyme activity was 38.61 U under optimal conditions. Over 50% of the enzyme activity was maintained under the optimal conditions after 9 h. Under the optimal conditions, the effect of metal ions on enzyme activity was analyzed. Ca2 ＋ , Fe2 ＋ and Li ~ slightly enhanced enzyme activity, while Mn2＋ and Co2＋ had little effect. Enzyme activity was modestly suppressed by Mg2~ , K~ and Na＋ , but considerably suppressed by Ag2~ and Zn2~ , with Cu2 ＋ showing the strongest inhibitory effects. [ Conclusion] A novel GM-degrading enzyme expressed by soil yeast was cloned, which can potentially be used in industrial applications to obtain eommereially useful guar gum-degradation products.
基金This work was supported by Grants from National High Technology Research and Development Program(No.2002AA2Z345B)and(No.2004AA2Z3803)of the Ministry of Science and Technology of China.
文摘To obtain human tissue inhibitor of metalloproteinase-2(TIMP-2)cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris,we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique,which was composed of frequently used codons in the highly expressed Pichia pastoris genes.Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing.The verified gene of TIMP2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2.The plasmid was transformed into GS115 cells of the methylotrophic yeast,Pichia pastoris by electroporation,and we got the expression cell through phenotype selection and induction with methanol.Separation,purification,and bioactivity analysis of the expressed products were performed.
基金Supported by the National Biotechnology Program for Crop Breeding(2013ZX08005004,2009ZX08009-089B)
文摘T4 lysozyme was engineered with disulfide bonds and expressed in Pichia pastoris. The secreted proteins were purified and made into powder by lyophilization. Recombinant protein purity was more than 70% measured by HPLC. The lytic activity of variant T4-lysozyme was measured by the lysis of the cell wall of Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Ralstonia solanacearum comb.nov, Clavibacter michiganensis subsp. michiganensis, X. campestris pv. malvacearum, Fusarium oxysporium sp. vasinfectum, Verticillium dahliae kleb. Inhibition zone assay showed that variant T4 lysozyme significantly inhibited X. o. oryzicola and X. c. malvacearum. The antifungal activities of this protein against F. o. vasinfectum and V. d. kleb were also analyzed.
基金supported by the National Key Research and Development Program of China(2019YFE0114600)National Natural Science Foundation of China(21776114).
文摘To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and expression.The biochemical and enzymatic properties of the new recombinant were characterized.As a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,respectively.Under the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation.To further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L bioreactors.Using high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L.In addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(min.mg),respectively.Thus,TrCel5A was effectively expressed in P.pastoris under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.
文摘利用含有强启动子 PAOX1 和 α-因子信号肽序列的巴斯德毕赤酵母载体 p PICZαA,构建出含牛白细胞介素 2(Bo IL - 2 )基因的重组质粒 Bo IL 2 - p PICZαA。线性化的重组表达载体转化到巴斯德毕赤酵母 X- 33及 KM71H中 ,筛选Zeocin高抗性酵母菌株 ,甲醇诱导目的蛋白表达。经 SDS- PAGE和 Western blot检测表明 ,Bo IL- 2以融合蛋白形式在胞内表达 ,但没能分泌到胞外。通过 Bo IL- 2在巴斯德毕赤酵母中的表达 ,重点讨论了信号肽。
