摘要
【目的】从毕赤酵母X-33的内源分泌蛋白中寻找高分泌能力的信号肽,以提高漆酶POXA1c的表达水平。【方法】通过二维电泳的数据分析,以及毕赤酵母的基因组测序结果筛选出7种毕赤酵母中高分泌水平的蛋白,利用其引导漆酶的分泌,通过测定漆酶的活力,来确定信号肽的分泌能力。【结果】七种信号肽都能引导漆酶的分泌,其中PHO5和FLO10信号肽,其引导下漆酶POXA1c的活力分别是自身信号肽引导下的2.5倍和2倍。组合信号肽FLO10-αpro和PHO5-αpro引导下漆酶的活力分别为自身信号肽引导下的3倍和3.5倍,分别比在α-MF引导下提高了20%和40%。【结论】毕赤酵母内源分泌蛋白的信号肽可以有效的引导外源蛋白的分泌,漆酶POXA1c在PHO5-αpro信号肽引导下,通过高密度发酵培养后,其漆酶活力达到57.98 U/m L。
[ Objective] Expression level of laccase POXAlc would be increased by screening effective signal peptide in Pichia pastoris. [ Methods] According to 2D-gel and profile of P. pastoris genome sequence, seven signal peptides from high secreted endogenous proteins of P. pastoris X-33 were chosen to evaluate their secreted ability by using POXAlc as reporter protein. [ Results] Compared with POXAlc＇ s native signal peptide, the signal peptide of repressible acid phosphatases PHO5 and leetin-like protein FLOIO showed 2.5-fold and 2-fold increase of laccase activity. Furthermore, PHO5-αpro and FLO10-αpro were constructed by fusing signal peptide of PHO5 and FLO10 with pro-peptide of α-MF respectively. The laeease activity under the leading of FLO10-αpro and PHO5-αpro showed 3-fold and 3.5- fold laccase activity higher than native signal peptide, and showed 20% and 40% increase compared with saccharomyces cerevisiae α- MF signal respectively. [ Conclusion] Signal peptides from high secreted endogenous proteins of P. pastoris X-33 could be effectively used to lead laccase expression in P. pastoris. The activity of POXA1c under the leading of the PHO5-αpro signal peptide was 57.98 U/mL after high density fermentation.
出处
《微生物学报》
CAS
CSCD
北大核心
2014年第12期1446-1452,共7页
Acta Microbiologica Sinica
基金
福建省产业化关键技术项目(闽财指[2010]358号)~~
关键词
毕赤酵母
信号肽
漆酶
Pichia pastoris, signal peptide, laccase
