Expression And Purification Of Serratia Marcescens Phospholipase A1 Fusion Protein

Phospholipase is a kind of enzyme that can hydrolyze glycerol phospholipids, there are phospholipase A1, A2, B, C, D and other subtypes. Among them, phospholipase A1 can specifically hydrolyze the Sn-1 acyl of natural phospholipids, and obtain L-beta lysophospholipids. The main purpose of phospholipase Al is the production of hemolysis phospholipid, soybean oil degumming, and the processing of yolk, cheese and the preparing of diglycerides etc. The advantages of high efficiency, low energy consumption and low pollution of phospholipase A1 have been widely concerned, researched and applied especially in the aspect of vegetable oil. Although the source of phospholipase A1 is widely, its pure enzyme have not been obtained so much, and is still far away from the requirements of industrial production, so a large number of production and purification of phospholipase A1 is necessary.Nowaday, with the rapid development of genetic engineering technology, the phospholipase A1 expressed in microbial expression systems has become a reality, but the soluble expression of a separate phospholipase A1 is low. Therefore, the phospholipase gene A1 which come from Serratia marcescens was cloned into different plasmids with different tags in this experiment, and the expression and purification of the fusion protein were also researched.The main aspects of the research in this paper were as following:(1) According to plaA gene sequence (ID:JX138535) in the GenBank and different plasmid vector, the specific primers were designed. The plaA gene from recombinant plasmid AP28 have obtained by PCR amplification, and recombinant plasmid pGEX-4T-2-A, pMAL-c2x-A and His-GST-A were constructed respectively. The recombinant plasmid was identified with enzyme digestion and sequencing. Results show that, the fusion tagging gene and plaA gene are in the same reading frame, indicating the successful construction of the three different tagged recombinant expression plasmid.(2) The recombinant plasmid pGEX-4T-2-A, pMAL-c2x-A and His-GST-A were sequenced correctly and transformed into Escherichia coli BL21(DE3) expression strains and then induced by IPTG at 37℃.The SDS-PAGE results showed that three different fusion protein GST-A, His-GST-A, MBP-A are properly express in the form of inclusion body, but the soluble protein was not detected. By reducing the induction temperature and IPTG concentration, the replacement of the culture media and host bacteria, as well as the replacement of vectors and other methods to promote the soluble expression of the fusion protein, but not yet detected soluble protein. Subsequently, the analysis on the sequence of phospholipase A1 gene from viscous Serratieae found that there is a strong hydrophobic peptide in 177-202 of the amino acid sequences, which may lead to fusion protein precipitate in the formation of inclusion bodies, and is aslo the reason why we can not obtainn soluble fusion protein.(3)The expression conditions of the expression strain BL21/His-GST-A were optimized, and the optimal conditions were as follows:the concentration of IPTG was 0.2 mmol/L, the temperature was 37 ℃, the strain density OD600nm was 0.5, and the induction time was 6 h. Under these conditions, the urea was used on the denaturation and renaturation of inclusion body, and then the renaturation solution was purified, the purified proteins were analysised by SDS-PAGE and the concentration of purified protein were measured by Bradford method. Finally, enzyme activity and enzymatic properties of the pure protein were studied. The results show that the purified His-GST-A protein was detected by SDS-PAGE as a single belt, and the protein concentration reached 293.59 ug/ml. The enzymatic properties was also studied, and the results show that under the optimum buffer of pH5.5 and the optimum temperature of 35℃, the enzyme activity reached 27.6U/mL, and the final specific enzyme activity was 94U/mg.(4) By comparing the specific activity of His-GST-A and His-A protein after the complex purification, the presence of GST protein tag exactly affect the activity of phospholipase A1, which decreased the enzyme activity of phospholipase A1. And about 2.53mg His-A protein freeze dried sample was obtained finally.In short, different tags were used to express soluble phospholipase A1 in this paper, but the results showed they have been expressed in inclusion body.Then, the fusion protein His-GST-A and His-A were by denaturation and renaturation from inclusion body, and the pure enzyme were also obtained. The effect of GST tag protein on the activity of phospholipase A1 was also compared. Finally, the His-A protein freeze dried sample was obtained, which lay the foundation for further study of phospholipase A1.