Institute of Agro-food Science and Technology, Chinese Academy of Agricultural Sciences (CAAS), Beijing, 100193, China.
Microb Cell Fact. 2018 Mar 28;17(1):50. doi: 10.1186/s12934-018-0894-y.
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.
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.
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.
已经在大肠杆菌中大量过表达了来自微生物来源的热稳定脂肪酶，然而，这些酶通常以低水平的酶活性和主要以包含体的形式产生。几项研究报告称，将重组蛋白的 N 端融合到信号肽上进行分泌生产已被用于解决该问题。一般来说，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要分泌的目标蛋白的类型。本研究旨在比较和优化信号肽，以有效分泌荧光假单胞菌 BJ-10 的耐热脂肪酶 lipBJ10。同时，在分泌可溶性和活性脂肪酶时，还进行了这种方法与细胞质分泌之间的比较研究。
使用 PelB 和五个天然大肠杆菌信号肽作为融合伴侣的融合表达产生了比非融合表达更多的可溶性和功能性重组 lipBJ10。将重组 lipBJ10 融合到这六种不同的信号肽中，可在大肠杆菌中分泌到周质中。所有融合表达的总脂肪酶活性均高于非融合表达。当 lipBJ10 融合到 DsbA 时，相对活性达到峰值，比非融合蛋白高 73.3 倍。当 DsbA 用作融合伴侣时，在形成包涵体最少的情况下获得了最高的活性（265.41 U/ml）；其他四种大肠杆菌信号肽在某种程度上导致了低活性和不溶性包涵体。因此，DsbA 是与 lipBJ10 融合以有效生产可溶性和功能性蛋白的最佳信号肽伴侣。
我们发现与这些信号肽融合，特别是与 DsbA 融合，与非融合 lipBJ10 相比，可显著减少包涵体的形成，并增强 lipBJ10 的功能和可溶性。我们在这里报告的结果可为其他脂肪酶的高水平表达提供参考，以实现可能的工业应用。
