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The Soluble Expression and Biological Activity Detection of Recombinant Mabinlin Ⅱ in E.Coli

Author: YaoJing
Tutor: GuoJianChun; HuXinWen
School: Hainan University
Course: Biochemistry and Molecular Biology
Keywords: Capparis masaikai L. Sweet protein Escherichia coli expression andpurification sweet-taste activity analysis
CLC: S188
Type: Master's thesis
Year: 2013
Downloads: 3
Quote: 0
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Abstract


Capparis masaikai L. is currently one of the seven sweet-protein-producing plants in word, which is a peculiar wild plant in China. Mabinlin II used as a a new type of sweetener has a broad application prospect when, because it possesses many advanced properties, such as the best heat stable and acid resistant, and higher sweet taste of being around400-fold sucrose on weight. However, the scarcity of the plant resources, make its application to be restricted in the food industry. In order to obtain more Mabinlin II protein resources and find the soluble expression form in vitro activity, in this study, the recombinant mabinlin II proteins were expressed and purified in the microorganism expression systems of Escherichia coli. The activity and thermal stability of mabinlin Ⅱ were further analyzed, and ultimately acquire recombinant mabinlin Ⅱ with highly purity, which will be experimental basis and technical support for researching and developing a new kind of sweetener on the basis of mabinlin Ⅱ.According to the structure characteristics of its sweet-taste activity, mabinlin Ⅱ gene was alternatively spliced and recombined to four kinds of recombinant mabinlin Ⅱ genes, in which mabinlin Ⅱ fused with two different fusion tag were expressed in Escherichia coli. The separation and purification systems of Escherichia coli for the expression of recombinant mabinlin Ⅱ proteins in form of soluble protein were established. Western-blot analysis was used to study the expression of recombinant mabinlin Ⅱ proteins. Furthermore, sweet-taste activity and thermal stability of the purified recombinant mabinlin Ⅱ proteins were identified. The conclusions were showed as follows:1. Mabinlin Ⅱ gene was spliced and recombined to four kinds of recombinant mabinlin Ⅱ genes(B、AB、AMB and MBL), then subcloned into pCold-SUMO and pGEX-4T-1vectors to result in eight recombined plasmids and transformed into E. coli BL21(DE3) host cells.2. After induction by IPTG, the pCold-sumo-B, pCold-sumo-AB and pCold-sumo-AMB were directly expressed in the form of soluble protein in E. coli expression system, while pCold-sumo-MBL failed to expressed. The pGEX-4T-B, pGEX-4T-AB, pGEX-4T-AMB and pGEX-4T-MBL were directly expressed in the form of inclusion body in E. coli expression system.3. The optimization of express conditions for the recombinant mabinlinⅡ pCold-sumo-B, pCold-sumo-AB, pCold-sumo-AMB were researched. The soluble protein yields of the recombinant mabinlinⅡ proteins were highest when the OD600of the cultured cells was0.6, the induction concentration of IPTG and lactose were0.6mmol/L and8mg/mL, and the incubating time after induction was24h at15℃.4. The recombinant mabinlin Ⅱ soluble proteins were purified by Ni-NTA affinity chromatography. The soluble proteins with highly purity were aquired, when the elution proteins were collected by using250mmol/L imidazole as elution solution.5. The purified soluble proteins SUMO-B, SUMO-AB and SUMO-AMB expressed in E. coli could be detected by anti-mabinlinⅡ polyclonal antibody immunodetection using Western blot. The purified soluble protein SUMO-B and SUMO-AMB tasted sweet, which was around100-fold sucrose sweetness on weight. Moreover, the soluble proteins SUMO-B and SUMO-AMB were thermal stability to some extent.In conclusion, the separation and purification systems for recombinant mabinlin Ⅱ soluble proteins expressed in E. coli were successfully set up. Evaluation systems of Western-blot identification, sweet-taste activity and thermal stability were also established for recombinant mabinlin Ⅱ soluble proteins. Recombinant proteins SUMO-B and SUMO-AMB with slight sweet-taste and certain thermal stability were successfully acquired in the E. coli expression system, which will be a technical support for mabinlinⅡ protein in other expression systems to research and development a new kind sweetener.

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