热带念珠菌β-葡聚糖合成酶KRE9基因原核表达及其比活力测定
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摘要
【目的】原核表达热带念珠菌(Candida tropicalis MYA-3404)β-葡聚糖合成酶(KRE9),并进行酶比活力测定,为研究其结构和生物学功能提供参考。【方法】提取热带念珠菌DNA,PCR扩增KRE9基因,构建其原核表达载体pET28a-KRE9后转化大肠杆菌(Escherichia coli)BL21(DE3)感受态细胞,通过IPTG诱导表达及组氨酸(His)标签纯化树脂亲和柱纯化获得融合蛋白KRE9,并利用3,5-二硝基水杨酸(DNS)显色法测定其比活力。【结果】克隆获得的KRE9基因全长816 bp,编码271个氨基酸,与参考基因序列(GenBank登录号XM_002547984)的相似性高达99.75%。将其原核表达载体pET28a-KRE9转化大肠杆菌BL21(DE3)感受态细胞,通过诱导表达及纯化获得重组蛋白KRE9,经SDS-PAGE检测,发现其纯度较高,大小约35 k D。Western blotting鉴定结果显示,重组蛋白KRE9能与抗His抗体发生特异性结合。重组蛋白KRE9比活力为9.169×10~4U/mg。【结论】重组蛋白KRE9具有良好的特异性和较高β-葡聚糖合成酶活性,可用于其结构和生物学功能研究。
【Objective】β-glucan synthase KRE9 of Candida tropicalis MYA-3404 was expressed and purified,and its specific activity was analyzed,in order to provide reference for the study on the structure and biological function of β-glucan synthase KRE9.【Method】The DNA of C. tropicalis was extracted,gene KRE9 was amplified by PCR,and prokaryotic expression vector p ET28 a-KRE9 was constructed. Competent cell of Escherichia coli BL21(DE3)was transfered.Fusion protein KRE9 was obtained by induction of IPTG as well as histidine(His)tag purificationof resin affinity chromatography,and its specific activity was determined by 3,5-dinitrosalicylic acid(DNS)colorimetricmethod.【Result】The cloned gene KRE9 was 816 bp in total length,encoding 271 amino acids. The sequencing results showed that gene KRE9 was highly similar to reference genes(GenBank accession number XM_002547984),which reached 99.75%. The prokaryotic expression vector p ET28 a-KRE9 was transformed into competent cell of E. coli BL21(DE3). Then the recombinant protein KRE9 was obtained by induction and purification. SDS-PAGE detection results showed that the recombinant proteins was 35 kDa with high purity.Western blotting identification indicated that there was specific binding between purified recombinant protein KRE9 and His antibody. The specific activity of KRE9 was 9.169×10~4 U/mg.【Conclusion】Recombinant protein vegetable proteinis of fine specificity and high β-glucan synthase activity,and it can be used for its structural and biological function research.
【Objective】β-glucan synthase KRE9 of Candida tropicalis MYA-3404 was expressed and purified,and its specific activity was analyzed,in order to provide reference for the study on the structure and biological function of β-glucan synthase KRE9.【Method】The DNA of C. tropicalis was extracted,gene KRE9 was amplified by PCR,and prokaryotic expression vector p ET28 a-KRE9 was constructed. Competent cell of Escherichia coli BL21(DE3)was transfered.Fusion protein KRE9 was obtained by induction of IPTG as well as histidine(His)tag purificationof resin affinity chromatography,and its specific activity was determined by 3,5-dinitrosalicylic acid(DNS)colorimetricmethod.【Result】The cloned gene KRE9 was 816 bp in total length,encoding 271 amino acids. The sequencing results showed that gene KRE9 was highly similar to reference genes(GenBank accession number XM_002547984),which reached 99.75%. The prokaryotic expression vector p ET28 a-KRE9 was transformed into competent cell of E. coli BL21(DE3). Then the recombinant protein KRE9 was obtained by induction and purification. SDS-PAGE detection results showed that the recombinant proteins was 35 kDa with high purity.Western blotting identification indicated that there was specific binding between purified recombinant protein KRE9 and His antibody. The specific activity of KRE9 was 9.169×10~4 U/mg.【Conclusion】Recombinant protein vegetable proteinis of fine specificity and high β-glucan synthase activity,and it can be used for its structural and biological function research.
引文
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张萍,赵强,魏东盛,杨娇,朱项阳,朱旭东.2016.酿酒酵母Snf1/AMPK蛋白激酶通过调节细胞壁合成相关基因的表达影响细胞壁完整性[J].微生物学报,56(7):1132-1140.[Zhang P,Zhao Q,Wei D S,Yang J,Zhu X Y,Zhu X D.2016.Snf1/AMPK affects cell wall integrity through regulating the transcription of cell wall as sembly-related genes in Saccharomyces cerevisiae[J].Microbiology China,56(7):1132-1140.]
周小玲,沈微,饶志明,王正祥,诸葛健.2004.一种快速提取真菌染色体DNA的方法[J].微生物学通报,31(4):89-92.[Zhou X L,Shen W,Rao Z M,Wang Z X,Zhuge J.2004.A rapid method for preparation of fungal chromosome DNA[J].Microbiology China,31(4):89-92.]
Amprayn K,Rose M T,Kecskés M,Pereg L,Nguyen H T,Kennedy I R.2012.Plant growth promoting characteristics of soil yeast(Candida tropicalis HY)and its effectiveness for promoting rice growth[J].Applied Soil Ecology,61(5):295-299.
Douglas C M,Marrinan J A,Li W,Kurtz M B.1994.A Saccharomyces cerevisiae mutant with echinocandin-resistant1,3-beta-D-glucan synthase[J].Journal of Bacteriology,176(18):5686-5696.
Gomaa A M,Moawad S S,Ebadah I M A,Salim H A.2005.Application of bio-organic farming and its influence on certain pests infestion,growth and productivity of potato plants[J].Research Journal of Applied Sciences,1(2):2005-2211.
He Z,Luo L,Keyhani N O,Yu X,Ying S,Zhang Y.2017.The C-terminal MIR-containing region in the Pmt1 O-mannosyltransferase restrains sporulation and is dispensable for virulence in Beauveria bassiana[J].Applied Microbiology&Biotechnology,101(3):1143-1161.
Lesage G,Bussey H.2006.Cell wall assembly in Saccharomyces cerevisiae[J].Microbiology and Molecular Biology Reviews,70(2):317-343.
Lussier M,Sdicu A M,Shahinian S,Bussey H.1998.The Candida albicans KRE9 gene is required for cell wallβ-1,6-glucan synthesis and is essential for growth on glucose[J].Proceedings of the National Academy of Sciences of the United States of America,95(17):9825-9830.
Mata D C.2013.Producción de la proteína recombinante Kre9 del hongo patógeno Candida albicans con actividad proinflamatoria y protumoral[D].Spain:Universidad del País Vasco.
Mekki B B,Ahmed A G.2005.Growth,yield and seed quality of soybean(Glycine max L)as affected by organic,biofertilizer and yeast application[J].Research Journal of Agriculture&Biological Sciences,1(4):320-324.
Mostert T T,Divol B.2014.Investigating the proteins released by yeasts in synthetic wine fermentations[J].International Journal of Food Microbiology,171(5):108-118.
Pan H P,Wang N,Tachikawa H,Nakanishi H,Gao X D.2017.β-1,6-glucan synthesis-associated genes are required for proper spore wall formation in Saccharomyces cerevisiae:Yeast spore wall andβ-1,6-glucan[J].Yeast,doi:10.1002/yea.3244.
Patle S,Lal B.2007.Ethanol production from hydrolysed agricultural wastes using mixed culture of Zymomonas mobilis and candida tropicalis[J].Biotechnology Letters,29(12):1839-1843.
Puppala K R,Naik T,Shaik A,Dastager S,Ravi K V,Khire J,Dharne M.2018.Evaluation of Candida tropicalis(NCIM3321)extracellular phytase having plant growth promoting potential and process development[J].Biocatalysis and Agricultural Biotechnology,13:225-235.
Radi?D S,Pavlovi?V P,Lazovi?M M,Jovi?i?-Petrovi?J P,Karli?i?V M,Lalevi?B T,Rai?evi?V B.2017.Coppertolerant yeasts:Raman spectroscopy in determination of bioaccumulation mechanism[J].Environmental Science&Pollution Research,24(27):21885-21893.
Weber L.2014.Characterization of Schizosachharomyces pombe Sup11p,a protein involved in beta-1,6-glucan bio synthesis[D].Heidelberg:Ruperto-Carola University of Heidelberg.
关波.2014.改良人血清白蛋白融合蛋白在毕赤酵母中分泌表达的研究[D].无锡:江南大学.[Guan B.2014.Improvement of secretory expression of human serum albumin fusion protein in Pichia pastoris[D].Wuxi:Jiangnan University.]
李鹏.2012.灰树花β-葡聚糖合成酶的提取、纯化及其酶学性质研究[D].郑州:河南工业大学.[Li P.2012.Extraction,purification and characterization of Grifola frondosaβ-glucan synthase[D].Zhengzhou:Henan University of Technology.]
陆志方.2016.固相合成抗菌肽CGA-N46及其衍生物生物信息学分析和生物活性研究[D].郑州:河南工业大学.[Lu Z F.2016.Study of solid phase synthesis of antifungal peptide CGA-N46 and its derivatives of bioinformatics analysis and biological activity[D].Zhengzhou:Henan University of Technology.]
汪家政,范明.2001.蛋白技术手册[M].北京:科学技术出版社.[Wang J Z,Fan M.2001.Handbook of Protein Technology[M].Beijing:Science and Technology Press.]
王靖瑶,王天女,卢磊,张帅,赵敏.2014.大肠杆菌I型分泌表达系统研究进展及提高蛋白表达量的策略[J].中国生物工程杂志,34(6):98-104.[Wang J Y,Wang T N,Lu L,Zhang S,Zhao M.2014.Research advances in secretary production of recombinant protein using Escherichia coli type I secretion systen and strategies for enhancement of secretion of type I pathway[J].Journal of Chinese Biotechnology,34(6):98-104.]
王卫国.2012.一种测定β-葡聚糖合成酶活力的方法:102443621B[P].2012-05-09.[Wang W G.2012.Method for detecting activity ofβ-glucan synthase:102443621B[P].2012-05-09.]
魏春红.2012.现代分子生物学实验技术[M].北京:高等教育出版社.[Wei C H.2012.Experiment Technology for Modern Molecular Biology[M].Beijing:Higher Education Press.]
薛雯雯.2010.CGA-N46基因在枯草芽孢杆菌中的多顺反子表达及表达条件优化[D].郑州:河南工业大学.[Xue W W.2010.Polycistronic expression of CGA-N46 gene in Bacillus subtilis and its fermentation optimization[D].Zhengzhou:Henan University of Technology.]
薛正莲,张珂.2013.表达重组谷氨酸脱羧酶工程菌发酵条件的优化[J].中国生物制品学杂志,26(1):100-104.[Xue Z L,Zhang K.2013.Optimization of fermentation condition of recombinant E.coli for expression of glutamate decarboxylase[J].Chinese Journal of Biologicals,26(1):100-104.]
赵丽洋.2006.白色念珠菌β-1,3-葡聚糖合成酶抑制剂筛选模型的建立及研究白色念珠菌β-1,3-葡聚糖合成酶调节亚基Ca Rho的克隆表达[D].北京:中国协和医科大学.[Zhao L P.2006.Establishment and study of a screening model forβ-1,3-glucan synthetase inhibitors of Candida albicans Cloning and expression of the regulatory subunit Ca Rho of Candida albicansβ-1,3-glucan synthase[D].Beijing:Peking Union Medical College.]
翟中会,陈希南,王娟.2008.利用Primer Premier 5.0进行引物设计[J].西北医学教育,16(4):695-698.[Zhai Z H,Chen X N,Wang J.2008.Primer design with primer premier 5.0[J].Northwestern Medical Education,16(4):695-698.]
张萍,赵强,魏东盛,杨娇,朱项阳,朱旭东.2016.酿酒酵母Snf1/AMPK蛋白激酶通过调节细胞壁合成相关基因的表达影响细胞壁完整性[J].微生物学报,56(7):1132-1140.[Zhang P,Zhao Q,Wei D S,Yang J,Zhu X Y,Zhu X D.2016.Snf1/AMPK affects cell wall integrity through regulating the transcription of cell wall as sembly-related genes in Saccharomyces cerevisiae[J].Microbiology China,56(7):1132-1140.]
周小玲,沈微,饶志明,王正祥,诸葛健.2004.一种快速提取真菌染色体DNA的方法[J].微生物学通报,31(4):89-92.[Zhou X L,Shen W,Rao Z M,Wang Z X,Zhuge J.2004.A rapid method for preparation of fungal chromosome DNA[J].Microbiology China,31(4):89-92.]
Amprayn K,Rose M T,Kecskés M,Pereg L,Nguyen H T,Kennedy I R.2012.Plant growth promoting characteristics of soil yeast(Candida tropicalis HY)and its effectiveness for promoting rice growth[J].Applied Soil Ecology,61(5):295-299.
Douglas C M,Marrinan J A,Li W,Kurtz M B.1994.A Saccharomyces cerevisiae mutant with echinocandin-resistant1,3-beta-D-glucan synthase[J].Journal of Bacteriology,176(18):5686-5696.
Gomaa A M,Moawad S S,Ebadah I M A,Salim H A.2005.Application of bio-organic farming and its influence on certain pests infestion,growth and productivity of potato plants[J].Research Journal of Applied Sciences,1(2):2005-2211.
He Z,Luo L,Keyhani N O,Yu X,Ying S,Zhang Y.2017.The C-terminal MIR-containing region in the Pmt1 O-mannosyltransferase restrains sporulation and is dispensable for virulence in Beauveria bassiana[J].Applied Microbiology&Biotechnology,101(3):1143-1161.
Lesage G,Bussey H.2006.Cell wall assembly in Saccharomyces cerevisiae[J].Microbiology and Molecular Biology Reviews,70(2):317-343.
Lussier M,Sdicu A M,Shahinian S,Bussey H.1998.The Candida albicans KRE9 gene is required for cell wallβ-1,6-glucan synthesis and is essential for growth on glucose[J].Proceedings of the National Academy of Sciences of the United States of America,95(17):9825-9830.
Mata D C.2013.Producción de la proteína recombinante Kre9 del hongo patógeno Candida albicans con actividad proinflamatoria y protumoral[D].Spain:Universidad del País Vasco.
Mekki B B,Ahmed A G.2005.Growth,yield and seed quality of soybean(Glycine max L)as affected by organic,biofertilizer and yeast application[J].Research Journal of Agriculture&Biological Sciences,1(4):320-324.
Mostert T T,Divol B.2014.Investigating the proteins released by yeasts in synthetic wine fermentations[J].International Journal of Food Microbiology,171(5):108-118.
Pan H P,Wang N,Tachikawa H,Nakanishi H,Gao X D.2017.β-1,6-glucan synthesis-associated genes are required for proper spore wall formation in Saccharomyces cerevisiae:Yeast spore wall andβ-1,6-glucan[J].Yeast,doi:10.1002/yea.3244.
Patle S,Lal B.2007.Ethanol production from hydrolysed agricultural wastes using mixed culture of Zymomonas mobilis and candida tropicalis[J].Biotechnology Letters,29(12):1839-1843.
Puppala K R,Naik T,Shaik A,Dastager S,Ravi K V,Khire J,Dharne M.2018.Evaluation of Candida tropicalis(NCIM3321)extracellular phytase having plant growth promoting potential and process development[J].Biocatalysis and Agricultural Biotechnology,13:225-235.
Radi?D S,Pavlovi?V P,Lazovi?M M,Jovi?i?-Petrovi?J P,Karli?i?V M,Lalevi?B T,Rai?evi?V B.2017.Coppertolerant yeasts:Raman spectroscopy in determination of bioaccumulation mechanism[J].Environmental Science&Pollution Research,24(27):21885-21893.
Weber L.2014.Characterization of Schizosachharomyces pombe Sup11p,a protein involved in beta-1,6-glucan bio synthesis[D].Heidelberg:Ruperto-Carola University of Heidelberg.