Enhancing Extracellular Expression Of Recombinant Proteins In Escherichia Coli With Co-expression Of Phospholipase C

In the Escherichia coli secretion system, the recombinant proteins with extracellular expression were secreted into culture medium, which could make the purification simple. In the previous studies, we found that the cutinase from Thermobifida fusca could hydrolyze the phospholipids in the membrane, which would enhance the cell membrane permeability, and then proteins in the cytoplasm were released into culture medium. But the hydrolysis of cutinase produced lysophospholipids which could give rise to lots of bubble. Lysophospholipids make the control of the production process diffiult. Bacillus cereus phospholipase C(PLC) has the similar hydrolysis reaction of phospholipids. In the speculation, PLC could also enhance recombinant proteins estracellular expression, and the productions of the hydrolysis would not lead to bubble.In this laoratory, PLC was co-expressed with model proteins, comparing the results of co-expressed model proteins and the model proteins without co-expression. The main results are as follows:1. The expression of PLC in E. coli: The Bacillus cereus genome was used as template for amplifying PLC gene by the polymerase chain reaction. After PCR, a 789-bp fragment was obtained. The recombinant plasmids, p ETDuet-1-plc, were constructed and transformed into E. coli BL21(DE3). The engineered cells were grown in TB medium. After 20 hours, the activity of PLC in culture supernatant was 14.2 U?m L-1, which was 95.3% of the total enzyme activity in culture supermatant and cytoplasm. At the end of the fermentation process, the cells expressing PLC exhibited irregular morphology but no obvious cell lysis, which was similar to T. fusca cutinase.2. Co-expression of PLC and extracellular model proteins: The three secretory enzymes, β-galactosidase(86 k Da), α-cyclodextrin glycosyltransferase(76 k Da) and xylanase(43 k Da) were co-expressed with PLC. In shake flask fermentation, the co-expressed β-galactosidase activity was similar to that without co-expression. The productivity of the co-expression system of α-cyclodextrin glycosyltransferase and xylanase was 2.7 and 4.0-fold that of the control without co-expression. It proves that the protein molecular mass was one of the factors which associate with effect of co-expression. When the protein molecular bigger than the scope, co-expression would not enhance its exthacellular secretion. And the smaller protein molecular would lead to a better exthacellular secretion. The co-expressed xylanase was purified. The specific activity, optimal temperature and p H were all similar to the xylanase without co-expression. Utilizing fed-batch strategy in 3 L fermentor of the recombinant xylanase, the co-expressed xylanase activity in culture supernatant was 678.6 U·m L-1, and the productivity was 21.7 U·m L-1·h-1. The activity of xylanase without co-expression in culture supernatant was 550.2 U·m L-1, and the productivity was 12.1 U·m L-1·h-1. The results showed that xylanase co-expressed with PLC has a favourable potential in industrial application.3. Co-expression of PLC and intracellular model proteins: The three secretory enzymes, isoamylase(79 k Da), trehalose synthase(66 k Da) and glucose isomerase(42 k Da) were co-expressed with PLC. In shake flask fermentation, the co-expressed isoamylase activity was similar to that without co-expression, which could only be detected within the cell. The trehalose synthase activity and glucose isomerase activity in culture supernatant was 35.2% and 92.7% of the total enzyme activity. It proves that the protein molecular mass was one of the factors which associate with effect of co-expression, which is similar to the co-expression of PLC and extracellular model proteins. The co-expressed glucose isomerase was purified. The specific activity, optimal temperature and p H were all similar to the glucose isomerase without co-expression. Utilizing fed-batch strategy in 3 L fermentor, the enzyme activity reached 17.7 U·m L-1 in 24 hours. The results showed that glucose isomerase co-expressed with PLC has a favourable potential in industrial application.