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Expression, Purificationin and Activity Assay in E.coli of a Novel Fusion Protein Cecropin B- Human Lysozyme

Author: WengZuo
Tutor: BianLiuJiao
School: Northwestern University
Course: Biochemical Engineering
Keywords: peptides Cecropins B human lysozyme Fusion protein Expression Purification Activity
CLC: R91
Type: Master's thesis
Year: 2007
Downloads: 244
Quote: 1
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Abstract


Anti-bacterial peptide (ABP) is a kind of small size molecular peptides encoded by a given gene, which is important for the host to defend pathogenic bacterial invaders. These peptides have broad spectrum of antibacterial effect and inhibit DNA replication of some virus. They can kill some cancer cells selectively. Insect cecropin has a specific chemical structure and has a new target point to bacterial cell membrane, which is different from other general antibiotic. It may destroy the cell membrane directly, and there is no toxic effect to animal and human body. While human lysozyme also have the antibacterial and antivirus activity. Human lysozyme is a protein secreted by human cell, so it is compatible with human body. When used in clinic, human lysozyme is safer than other lysozyme and has no irritations and side effects. In this research, we constructed Cecropin B- human lysozyme engineering bacteria, then studied on the conditions of expression and purification .And at last we expressed the fusion protein with more effective antibacterial ability.ABP and lysozyme have strong anti-spectrum antibacterial activity, which caused much inconvenience for recombinant protein expression. Therefore, we used BL21 (DE3) pLysS strain to express recombinant protein CB-hLy in two-stage. The first stage is for plasmid growth, in which bacteria was cultured at 37℃. It is to ensure stability and enhance plasmid copy number. The next stage is to express the recombinant protein. After saturation in cell growth, we induced recombinant protein CB-hLy by low temperature of 25℃. In addition to the inspection of temperature, we also studied the induced factors such as medium and induction time. After bacteria grow at 37℃in TB medium, the same volume of fresh TB medium is replaced, and then induced for 4h at 25℃. The experimental results show that the supernatant protein expression could almost get to 40%. AS the protein with a 6×His, we use nickel metal ion affinity chromatography to purify the protein in first step. By sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) analysis showed that the purity is about 85% and protein yield is 1.57%. After the first step, the protein is repurified by gel exclusion chromatography. Finally, the protein purity get to 94% , and protein yield is 0.49%. Then we determined biological activity of the product by agar osmosis. The results show that the purified product of a good inhibition of Staphylococcus aureus.

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