高效毕赤酵母表达载体的改造与应用
|
摘要
构建一种可促进外源蛋白在毕赤酵母中高表达的载体,实现在同一载体中外源基因与二硫键异构酶共表达,同时失活YPS1基因。以p PICZαA载体为骨架载体,将酵母YPS1基因C端和N端非功能区序列连接至p PICZαA载体,获得重组载体p PICZαA-YPSΔ;将酵母PDI基因序列连接至p PICZαA载体,获得重组载体p PICZαAPDI,再以重组载体p PICZαA-PDI为模板通过PCR获得PDI表达盒,然后将PDI表达盒连接至重组载体p PICZαAYPSΔ,筛选获得高效表达载体p PICZαA-PDI-YPSΔ。最后,将外源基因HSA、h GH导入此载体,转化毕赤酵母GS115,检测HSA和h GH蛋白的表达。结果显示,高效表达载体p PICZαA-PDI-YPSΔ构建成功,外源蛋白HSA、h GH利用此载体,可获得高表达,具有很好的工业前景。
To construct a vector for promoting the high expression of exogenous protein in Pichia pastoris,the exogenous gene was coexpressed with PDI in the same vector,and the YPS1 gene was inactivated simultaneously. Using p PICZαA as backbone vector,the C and N-terminal nonfunctional area sequence of YPS1 pichia gene was connected to p PICZαA vector,then the recombinant vector p PICZαA-YPSΔ was obtained. When pichia PDI gene sequence was connected to p PICZαA vector,recombinant vector p PICZαA-PDI was produced. Next,PDI expression cassette was gained using PCR method with recombinant vector p PICZαA-PDI as a template. After connecting PDI expression cassette to recombinant vector p PICZαA-YPSΔ,the high expression vector p PICZαA-PDI-YPSΔ was obtained via screening. Finally,the exogenous genes HSA and h GH were introduced to the vector,then converting to Pichia GS115 in order to test the expression of HSA and h GH. The result implied that high expression vector p PICZαA-PDI-YPSΔ was built successfully.The exogenous gene HSA and h GH achieved high expression with this vector. High expression vector p PICZαA-PDI-YPSΔ could be effectively applied to industrial applications.
To construct a vector for promoting the high expression of exogenous protein in Pichia pastoris,the exogenous gene was coexpressed with PDI in the same vector,and the YPS1 gene was inactivated simultaneously. Using p PICZαA as backbone vector,the C and N-terminal nonfunctional area sequence of YPS1 pichia gene was connected to p PICZαA vector,then the recombinant vector p PICZαA-YPSΔ was obtained. When pichia PDI gene sequence was connected to p PICZαA vector,recombinant vector p PICZαA-PDI was produced. Next,PDI expression cassette was gained using PCR method with recombinant vector p PICZαA-PDI as a template. After connecting PDI expression cassette to recombinant vector p PICZαA-YPSΔ,the high expression vector p PICZαA-PDI-YPSΔ was obtained via screening. Finally,the exogenous genes HSA and h GH were introduced to the vector,then converting to Pichia GS115 in order to test the expression of HSA and h GH. The result implied that high expression vector p PICZαA-PDI-YPSΔ was built successfully.The exogenous gene HSA and h GH achieved high expression with this vector. High expression vector p PICZαA-PDI-YPSΔ could be effectively applied to industrial applications.
引文
[1]武婕,张晓雪,余河水,等.毕赤酵母工程菌高密度发酵研究与进展[J].中国生物工程杂志,2016,36(1):108-114.
[2]王庆华,高丽丽,梁会超,等.影响毕赤酵母高效表达重组蛋白的主要因素及其研究进展[J].药学学报,2014(12):1644-1649.
[3]WERTEN M W,VAN DEN BOSCH T J,WIND R D,et al. High-yield secretion of recombinant gelatins by Pichia pastoris[J]. Yeast,1999,15(11):1087-1096.
[4]HOHENBLUM H,BORTH N,MATTANOVICH D. Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry[J]. J Biotechnol,2003,102(3):281-290.
[5]卢临萍.二硫键异构酶过表达对家蚕乙酰胆碱酯酶在毕赤酵母中表达的作用研究[D].广州:华南农业大学,2016.
[6]INAN M,ARYASOMAYAJULA D,SINHA J,et al. Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase[J]. Biotechnol Bioeng,2006,93(4):771-778.
[7]秦作先,刘志敏. yapsin蛋白酶家族研究进展[J].生物技术通讯,2008,19(4):591-596.
[8]EGEL-MITANI M,ANDERSEN A S,DIERS II,et al. Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains[J]. Enzyme Microb Technol,2000,26(9/10):671-677.
[9]WERTEN M W,DE WOLF F A. Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2disruptant[J]. Appl Environ Microbiol,2005,71(5):2310-2317.
[10]龚香艺,丁重阳,刘立明,等.一种增加重组毕赤酵母拷贝数提高胰岛素前体产量的策略[J].微生物学报,2013,53(6):545-552.
[11]郭敏,雷清,刘晓,等.戊型肝炎病毒多拷贝分泌表达载体的构建及在毕赤酵母中的表达[J].中国新药杂志,2015,24(3):271-275.
[12]WILKINSON B,GILBERT H F. Protein disulfide isomerase[J]. Biochimica et Biophysica Acta,2004,1699(1/2):35-44.
[13]WANG Q H,LIANG L,LIU W C,et al. Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating genedosage,coexpressing with chaperones and fermenting in fed-batch mode[J]. J Asian Nat Prod Res,2017,19(6):581-594.
[14]YAO X Q,ZHAO H L,XUE C,et al. Degradation of HSA-AX15(R13K)when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast[J]. J Biotechnol,2009,139(2):131-136.
[2]王庆华,高丽丽,梁会超,等.影响毕赤酵母高效表达重组蛋白的主要因素及其研究进展[J].药学学报,2014(12):1644-1649.
[3]WERTEN M W,VAN DEN BOSCH T J,WIND R D,et al. High-yield secretion of recombinant gelatins by Pichia pastoris[J]. Yeast,1999,15(11):1087-1096.
[4]HOHENBLUM H,BORTH N,MATTANOVICH D. Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry[J]. J Biotechnol,2003,102(3):281-290.
[5]卢临萍.二硫键异构酶过表达对家蚕乙酰胆碱酯酶在毕赤酵母中表达的作用研究[D].广州:华南农业大学,2016.
[6]INAN M,ARYASOMAYAJULA D,SINHA J,et al. Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase[J]. Biotechnol Bioeng,2006,93(4):771-778.
[7]秦作先,刘志敏. yapsin蛋白酶家族研究进展[J].生物技术通讯,2008,19(4):591-596.
[8]EGEL-MITANI M,ANDERSEN A S,DIERS II,et al. Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains[J]. Enzyme Microb Technol,2000,26(9/10):671-677.
[9]WERTEN M W,DE WOLF F A. Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2disruptant[J]. Appl Environ Microbiol,2005,71(5):2310-2317.
[10]龚香艺,丁重阳,刘立明,等.一种增加重组毕赤酵母拷贝数提高胰岛素前体产量的策略[J].微生物学报,2013,53(6):545-552.
[11]郭敏,雷清,刘晓,等.戊型肝炎病毒多拷贝分泌表达载体的构建及在毕赤酵母中的表达[J].中国新药杂志,2015,24(3):271-275.
[12]WILKINSON B,GILBERT H F. Protein disulfide isomerase[J]. Biochimica et Biophysica Acta,2004,1699(1/2):35-44.
[13]WANG Q H,LIANG L,LIU W C,et al. Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating genedosage,coexpressing with chaperones and fermenting in fed-batch mode[J]. J Asian Nat Prod Res,2017,19(6):581-594.
[14]YAO X Q,ZHAO H L,XUE C,et al. Degradation of HSA-AX15(R13K)when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast[J]. J Biotechnol,2009,139(2):131-136.