从枯草芽孢杆菌1A 747菌株基因组克隆Am yX基因信号肽序列,构建枯草芽孢杆菌分泌表达载体pYGAm yX,转化枯草杆菌W B 700得W B 700(pYGAm yX)。采用重叠PCR技术,将Am yX基因信号肽突变,使其N端正电荷增加,H区疏水性降低,利用突变后信号...从枯草芽孢杆菌1A 747菌株基因组克隆Am yX基因信号肽序列,构建枯草芽孢杆菌分泌表达载体pYGAm yX,转化枯草杆菌W B 700得W B 700(pYGAm yX)。采用重叠PCR技术,将Am yX基因信号肽突变,使其N端正电荷增加,H区疏水性降低,利用突变后信号肽序列构建枯草杆菌分泌表达载体pYGMUT,转化枯草杆菌W B 700得W B 700(pYGMUT)。对W B 700(pYGAm yX)和W B 700(pYGMUT)的表达产物进行检测,研究Am yX基因信号肽突变对枯草杆菌蛋白分泌途径的影响。结果表明,W B 700(pYGAm yX)和W B 700(pYGMUT均能表达β-半乳糖苷酶,W B 700(pYGAm yX)的最高胞外酶活性达2 300 U,W B 700(pYGMUT)的最高酶活性达5 200 U。结果表明Am yX基因信号肽突变后构建的表达载体pYGMUT分泌表达β-半乳糖苷酶明显提高,说明β-半乳糖苷酶通过枯草杆菌T at途径分泌表达。展开更多
In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A,...In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.展开更多
文摘从枯草芽孢杆菌1A 747菌株基因组克隆Am yX基因信号肽序列,构建枯草芽孢杆菌分泌表达载体pYGAm yX,转化枯草杆菌W B 700得W B 700(pYGAm yX)。采用重叠PCR技术,将Am yX基因信号肽突变,使其N端正电荷增加,H区疏水性降低,利用突变后信号肽序列构建枯草杆菌分泌表达载体pYGMUT,转化枯草杆菌W B 700得W B 700(pYGMUT)。对W B 700(pYGAm yX)和W B 700(pYGMUT)的表达产物进行检测,研究Am yX基因信号肽突变对枯草杆菌蛋白分泌途径的影响。结果表明,W B 700(pYGAm yX)和W B 700(pYGMUT均能表达β-半乳糖苷酶,W B 700(pYGAm yX)的最高胞外酶活性达2 300 U,W B 700(pYGMUT)的最高酶活性达5 200 U。结果表明Am yX基因信号肽突变后构建的表达载体pYGMUT分泌表达β-半乳糖苷酶明显提高,说明β-半乳糖苷酶通过枯草杆菌T at途径分泌表达。
基金Project(13JJ9002)supported by Hunan Provincial Natural Science Foundation of ChinaProject(2012XK4081)supported by the Key Science Technology Plan Project of Hunan Provincial Science&Technology Department,ChinaProject(CX2012B124)supported by the Graduate Degree Thesis Innovation Program of Hunan Province,China
文摘In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.
