Recombinant Engineering-mediated Cell Self-shearing Systems, And Genomic-based N-acetyl-D-neuraminic Acid

Heterologous protein overexpressed in E. coli often forms insoluble inclusion bodies which complicates further functional study gene and protein. The maltose binding protein (MBP), a small molecule protein ubiquitin-like modifier (SUMO), bacterial translation initiation factor (IF2), nitrogen use substance A (NusA) and glutathione S-transferase (GST) tag can increase the soluble expression of the protein of interest. Solid metal affinity chromatography to purify histidine-tagged protein of interest is a highly effective protein purification technology.However, to study the structure and function of the protein labeling target proteins, it is necessary to separate the target protein fusion tags Representative method is the use of TEV protease cleavage of the fusion protein proteolytic enzyme sites to give the target protein, the purified target protein finally obtained by affinity chromatography. However, in many cases, the intracellular cleavage of the fusion protein is very important, not only to save time but also to optimize the experimental procedure. Co-expression of proteolytic enzymes within the cell and fusion protein can be purified before fusion protein tag and the target protein separately, to achieve self-splicing within the cell.The main aim about this topic is to fusion Beta tag with GFP, hRnBP and Slr 1975, respectively. From this research we understood that Beta can improve the GFP expression of soluble protein, what’s more, Beta can increased hRnBP and slr 1975 soluble protein, about 70%, but the difference about increment between hRnBP and slr 1975 are not so large. According to above experimental results, if we use Ni-NTA His tag affinity chromatography method to purify His-Beta-GFP, His-Beta-hRnBP and His-Beta-Slrl975 fusion protein, we can’t get satisfying final result.In this study, MBP-GFP fusion clone pLS3011 and MBP-TIMP clones were constructed and transferred to express the strain LS2416 TEV protease, complete intracellular self-splicing, and finally the use of His tag protein was purified by affinity chromatography. SDS-PAGE diagram showed high efficiency TEV cleavage enzyme, was almost 100%. Affinity chromatography was used to obtain a single GFP and N-TIMP protein bands.In this study, the genomic and plasmid levels explored the enzyme catalytic efficiency of Neu5Ac, and measured its yield by TBA method and the HPLC method. It showed a plasmid level is 41.3% and genomic level is 37.5%, compared the previous two-enzyme conjugate, although the conversion rate is lower, but the realization of the BT0453 and nanA level co-expression, shortening the distance between the two enzymes space, improves the intermediate ManNAc utilization, saves time, promote economic efficiency. Chromosome-based gene expression can also overcome the shortcomings of plasmid instability, metabolic burden, and the use of antibiotics.In this study, in order to improve the yield of Neu5Ac in LS2402 (BT0453 and nanA co-expression in the chromosome), by recombinant engineering, and I-Scel double strand break repair gene knockdown chloramphenicol cat, because the expression of this gene affects the Neu5Ac aldehyde reduced expression of the enzyme and isomerase. The engineered strain LS2802 was obtained in which no cat gene expression was observed from SDS-PAGE analysis.In order to further improve the Neu5Ac production, pLS2812 and pLS2814, which contain glucose-6-P-N-acetyltransferase (ScGNA1) and glucosamine synthase gene (glmS* 72) gene, were constructed, ScGNAl and glmS* 72 will be used for Neu5Ac producing strain knock-in, which will be helpful the chromosome based Neu5Ac biocatalysis.