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( Zu Yong He ),( Yuankai Huang ),( Yufeng Qin ),( Zhiguo Liu ),( Delin Mo ),( Peiqing Cong ),( Yaosheng Chen ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.4
The secretory efficiency of recombinant xylanase xynB from yeast Pichia pastoris between the α-factor preprosequence and a classical mammalian signal peptide derived from bovine β-casein was compared. The results showed that although the bovine β-casein signal peptide could direct highlevel secretion of recombinant xylanase, it was relatively less efficient than the α-factor preprosequence. In contrast, the bovine β-casein signal peptide caused remarkably more recombinant xylanase trapped intracellularly. Realtime RT-PCR analysis indicated that the difference in the secretory level between the two signal sequences was not due to the difference in the transcriptional efficiency.
( Huang Yuan Kai ),( Yao Sheng Chen ),( De Lin Mo ),( Pei Qing Cong ),( Zu Yong He ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.3
Xylanase has been used extensively in the industrial and agricultural fields. However, the low-yield production of xylanase from native species cannot meet the increasing demand of the market. Therefore, improving the heterologous expression of xylanase through basic gene optimization may help to overcome the shortage. In this study, we synthesized a high-GC-content native sequence of the thermostable xylanase gene xynB from Streptomyces olivaceoviridis A1 and, also designed a slightly AT-biased sequence with codons completely optimized to be favorable to Pichia pastoris. The comparison of the sequences` expression efficiencies in P. pastoris X33 was determined through the detection of single-copy-number integrants, which were quantified using qPCR. Surprisingly, the high GC content did not appear to be detrimental to the heterologous expression of xynB in yeast, whereas the optimized sequence, with its extremely skewed codon usage, exhibited more abundant accumulation of synthesized recombinant proteins in the yeast cell, but an approximately 30% reduction of the secretion level, deduced from the enzymatic activity assay. In this study, we developed a more accurate method for comparing the expression levels of individual yeast transformants. Moreover, our results provide a practical example for further investigation of what constitutes a rational design strategy for a heterologously expressed and secreted protein.
( Hai Long Liu ),( Yu Feng Qin ),( Yuan Kai Huang ),( Yao Sheng Chen ),( Pei Qing Cong ),( Zu Yong He ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.2
Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the α-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression.