Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide
来源期刊：中南大学学报(英文版)2015年第6期
论文作者：WANG Ye ZHENG Jia LIN Fu-lai ZHOU Hong-bo
文章页码：2088 - 2095
Key words：endo-1,4-β-mannosidase; pichia pastoris (p.pastoris); signal peptide; optimization
Abstract: 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, 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/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.
WANG Ye(王冶)1, ZHENG Jia(郑甲)1, LIN Fu-lai(林福来)1, ZHOU Hong-bo(周洪波)1, 2
(1. School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China;
2. Key Laboratory of Biometallurgy of Ministry of Education (Central South University), Changsha 410083, China)
Abstract: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, 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/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.
Key words:endo-1,4-β-mannosidase; pichia pastoris (p.pastoris); signal peptide; optimization
