利用非经典分泌蛋白质实现脂肪酶A的分泌表达

Effect of non-classical secreted proteins on Lipase A secretion

【目的】以枯草芽孢杆菌脂肪酶A(Lipase A)为报告蛋白,尝试利用4种非经典分泌蛋白质及其前50个氨基酸作为分泌信号以实现其分泌表达。【方法】我们扩增了脂肪酶A的编码基因和非经典分泌蛋白质的编码序列,构建了8种针对脂肪酶A的分泌表达载体,并转化至枯草芽孢杆菌WB800菌株,通过测定重组菌株的酶活、利用蛋白质电泳和免疫印迹等技术检测脂肪酶A的分泌情况【结果】以Pdh A的氨基酸序列和Sod A、Eno的前50氨基酸序列作为分泌信号的重组菌株较好的实现了脂肪酶A的分泌表达。【结论】部分非经分泌蛋白质的编码基因或其前50个氨基酸序列能够引导脂肪酶A分泌至细胞外。

[Objective] We used 50-amino acid-long peptides from the N-terminus of 4 different non-classically secreted proteins to study the secretion efficiency of Bacillus subtilis Lipase A via non-classical secretion pathway. [Methods] We amplified the coding sequences（ CDs） of Lipase A and N-terminus of non-classically secreted proteins,constructed 8fusion protein expression vectors containing both Lipase A CD and different secretion signal peptide and transformed them into B. subtilis WB800. Secretion efficiency of these fusion proteins was analyzed by enzyme activity,SDS-PAGE and Western-Blot. [Results] Recombinant Lipase A containing coding sequences of Pdh A or N-terminus of Sod A and Eno as secretion signals was efficiently secreted. [Conclusion] Parts of non-classically secreted proteins or N-terminus（ 50 amino acids） could guide Lipase A protein secretion.