PDF(Pubmed)
Abstract：
BACKGROUND: Thermostable lipases from microbial sources have been substantially overexpressed in E. coli, however, these enzymes are often produced with low-level enzymatic activity and mainly in the form of inclusion bodies. Several studies have reported that the secretory production of recombinant proteins fused their N-terminus to a signal peptide has been employed to resolve the problem. In general, the feasibility of this approach largely depends on the secretory pathway of signal peptide and the type of target protein to be secreted. This study was performed to compare and optimize signal peptides for efficient secretion of thermostable lipase lipBJ10 from Pseudomonas fluorescens BJ-10. Meanwhile, a comparative study between this method and cytoplasmic secretion was implemented in secreting soluble and active lipases.
RESULTS: Fusion expression using six signal peptides, i.e., PelB and five native E. coli signal peptides, as fusion partners produced more soluble and functional recombinant lipBJ10 than non-fusion expression. Recombinant lipBJ10, fused to these six diverse signal peptides, was secreted into the periplasm in E. coli. The total lipase activity in all cases of fusion expression was higher than those in non-fusion expression. The relative activity peaked when lipBJ10 was fused to DsbA, yielding a value 73.3 times greater than that of the non-fusion protein. When DsbA was used as the fusion partner, the highest activity (265.41 U/ml) was achieved with the least formation of inclusion bodies; the other four E. coli signal peptides, to some extent, led to low activity and insoluble inclusion bodies. Therefore, DsbA is the optimal signal peptide partner to fuse with lipBJ10 to efficiently produce soluble and functional protein.
CONCLUSIONS: We found that fusing to these signal peptides, especially that of DsbA, can significantly decrease the formation of inclusion bodies and enhance the function and solubility of lipBJ10 compared to non-fusion lipBJ10. Our results reported here can provide a reference for the high-level expression of other lipases with respect to a possible industrial application.
RESULTS: Fusion expression using six signal peptides, i.e., PelB and five native E. coli signal peptides, as fusion partners produced more soluble and functional recombinant lipBJ10 than non-fusion expression. Recombinant lipBJ10, fused to these six diverse signal peptides, was secreted into the periplasm in E. coli. The total lipase activity in all cases of fusion expression was higher than those in non-fusion expression. The relative activity peaked when lipBJ10 was fused to DsbA, yielding a value 73.3 times greater than that of the non-fusion protein. When DsbA was used as the fusion partner, the highest activity (265.41 U/ml) was achieved with the least formation of inclusion bodies; the other four E. coli signal peptides, to some extent, led to low activity and insoluble inclusion bodies. Therefore, DsbA is the optimal signal peptide partner to fuse with lipBJ10 to efficiently produce soluble and functional protein.
CONCLUSIONS: We found that fusing to these signal peptides, especially that of DsbA, can significantly decrease the formation of inclusion bodies and enhance the function and solubility of lipBJ10 compared to non-fusion lipBJ10. Our results reported here can provide a reference for the high-level expression of other lipases with respect to a possible industrial application.
摘要：
背景：来自微生物来源的热稳定脂肪酶已在大肠杆菌中大量过表达，然而,这些酶通常具有低水平的酶活性，主要以包涵体的形式产生。一些研究已经报道,分泌生产将其N-末端融合至信号肽的重组蛋白已经用于解决该问题。总的来说,这种方法的可行性在很大程度上取决于信号肽的分泌途径和待分泌的靶蛋白的类型。进行这项研究是为了比较和优化信号肽，以从荧光假单胞菌BJ-10有效分泌热稳定脂肪酶脂肪BJ10。同时,在分泌可溶性和活性脂肪酶时，对该方法与细胞质分泌进行了比较研究。
结果：使用六个信号肽的融合表达，即，PelB和五种天然大肠杆菌信号肽，作为融合伴侣比非融合表达产生更多的可溶性和功能性重组lipBJ10。重组lipBJ10，与这六种不同的信号肽融合，被分泌到大肠杆菌的周质中。在所有融合表达的情况下，总脂肪酶活性均高于非融合表达。当lipBJ10与DsbA融合时，相对活性达到峰值，产生的值是非融合蛋白的73.3倍。当DsbA用作融合伴侣时，活性最高(265.41U/ml),包涵体形成最少;其他四种大肠杆菌信号肽,在某种程度上,导致低活性和不溶性包涵体。因此,DsbA是与lipBJ10融合以有效产生可溶性和功能性蛋白质的最佳信号肽伴侣。
结论：我们发现与这些信号肽融合，尤其是DsbA,与未融合的lipBJ10相比，可以显着减少包涵体的形成并增强lipBJ10的功能和溶解度。我们在此报告的结果可为其他脂肪酶的高水平表达提供可能的工业应用参考。
结果：使用六个信号肽的融合表达，即，PelB和五种天然大肠杆菌信号肽，作为融合伴侣比非融合表达产生更多的可溶性和功能性重组lipBJ10。重组lipBJ10，与这六种不同的信号肽融合，被分泌到大肠杆菌的周质中。在所有融合表达的情况下，总脂肪酶活性均高于非融合表达。当lipBJ10与DsbA融合时，相对活性达到峰值，产生的值是非融合蛋白的73.3倍。当DsbA用作融合伴侣时，活性最高(265.41U/ml),包涵体形成最少;其他四种大肠杆菌信号肽,在某种程度上,导致低活性和不溶性包涵体。因此,DsbA是与lipBJ10融合以有效产生可溶性和功能性蛋白质的最佳信号肽伴侣。
结论：我们发现与这些信号肽融合，尤其是DsbA,与未融合的lipBJ10相比，可以显着减少包涵体的形成并增强lipBJ10的功能和溶解度。我们在此报告的结果可为其他脂肪酶的高水平表达提供可能的工业应用参考。
