不同信号肽对毕赤酵母表达漆酶的影响

Effect of signal peptides on the expression of laccase in Pichia pastoris

摘要:

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/mL。

Abstract:

Abstract:［Objective］Expression level of laccase POXA1c 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 POXA1c as reporter protein.［Results］ Compared with POXA1c’s native signal peptide，the signal peptide of repressible acid phosphatases PHO5 and lectin-like protein FLO10 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 laccase 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.