Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide
Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide
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DOI：
10.1007/s11771-015-2733-1
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发表时间：
2015-06
影响因子：
4.4
通讯作者：
Ye Wang;Jia Zheng;Fu-lai Lin;Hong-bo Zhou
中科院分区：
文献类型：
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作者：
Ye Wang;Jia Zheng;Fu-lai Lin;Hong-bo Zhou
为了提高重组毕赤酵母胞外内-1,4-β-甘露糖苷酶（MAN）的活性，对信号肽进行了优化研究。首先选择5个潜在的信号肽（W1、MF4I、INU1A、αpre、HFBI），通过SignalP 4.0进行分析，其中设计了W1。然后，将表达载体pgapz - α a中广泛使用的信号肽α-因子替换为这5个信号肽，重构5个新的表达载体。将表达载体转化为毕赤酵母后检测MAN活性。结果表明，与α-因子相比，W1、MF4I、INU1A、apre和HFBI信号肽的相对效率分别为23.5%、203.5%、0、79.7%和120.3%。进一步通过实时荧光定量PCR测定基因拷贝数，α-因子在1 ~ 6个基因拷贝数水平上介导的MAN活性分别为12.95、43.33、126.63、173.53、103.23和88.63 U/mL，而MF4I介导的MAN活性分别为79.22、133.89、260.14、347.5、206.15和181.89 U/mL。MF4I介导4个基因拷贝，MAN活性最高可达347.5 U/mL。这些结果表明，用MF4I替代信号肽α-因子，并将MAN基因拷贝数增加到适当数量，可以显著提高MAN的分泌表达。
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, MF4I, INU1A, αpre, HFBI) were chosen to be analyzed by SignalP 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector pGAPZαA 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, MF4I, INU1A, apre, 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/mL, while those mediated by MF4I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/mL, respectively. The maximum MAN activity reached 347.5 U/mL with 4 gene copies mediated by MF4I. These results indicate that replacing the signal peptide α-factor with MF4I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.