| Capparis masaikai Levl. a kind of peculiar wild plant in China, is rich in sweet protein mabinin in seeds.In five homologs of the mabinlin family, mabinlin Ⅱ possesses many advanced properties, such as being around400-fold sweeter than sucrose on weight basis, the most heat stable and acid resistant among seven types of plant sweet proteins. Thus it will be a novel natural sweetener in food industry and have broad market prospect. However, mabinlin Ⅱ could not be abundantly obtained from the C. masaikai plant as it is increasingly scarce, which is limited the use of naturally acquired mabinlin Ⅱ in the food additive industry. In this study, for expanding the protein resources and finding the active expression forms of mabinlin Ⅱ, recombinant mabinlin Ⅱ proteins were expressed and purified in two kinds of microorganism expression systems of Escherichia coli and Lactococcus lactis. The active structure forms of recombinant mabinlin Ⅱ were analyzed on this basis, the technique of acquiring recombinant mabinlin Ⅱ with highly purity in quantity and quickly isolation have been established, which will be foundation and technical support for researching and developing a new practical sweetener mabinlin Ⅱ.According to the structure characteristics of its sweet-taste activity, mabinlin Ⅱ gene was alternatively spliced and recombined to several kinds of recombinant mabinlin Ⅱ genes, and transformed into E. coli and food-grade L. lactis expression system. The separation and purification systems for the expressed recombinant mabinlin Ⅱ proteins in form of inclusion body and fused protein in E. coli were established; and the food-grade inducible expression systems of recombinant mabinlin Ⅱ proteins in form of secretive and intracellular expressions in L. lactis were also established. In addition, the mabinlin Ⅱ was extracted and purified from the seeds of C. masaikai, and anti-mabinlin Ⅱ polyclonal antibody was prepared by immunizing rabbits with purified natural mabinlin Ⅱ and purified by antigen-affinity chromatography. Western-blot analysis of the expressed recombinant mabinlin Ⅱ proteins in E. coli and L. lactis were studied. Furthermore, MALDI-TOF-TOF, heat stability and sweet-taste activity of the expressed recombinant mabinlin II proteins were identified. The main research results were showed as follows:1. Natural mabinlin Ⅱ proteins were extracted and purified from the seeds of C. masaikai by carboxymethyl cellulose chromatography, and the yield and purity were9.82%and86.9%, respectively. Anti-mabinlin Ⅱ polyclonal antibody was prepared by immunizing rabbits with purified mabinlin Ⅱ and purified by antigen-affinity chromatography. The titer of the antibody reached up to1:256000. Western-blot analysis indicated that anti-mabinlin Ⅱ polyclonal antibody could indentify both the natural and expressed recombinant mabinlin Ⅱ.2. Recombinant mabinlin Ⅱ genes MBL-BH, MBL-ABH and MBL-AXBH were directly expressed in the form of inclusion body in E. coli expression system. The inclusion body yields of recombinant mabinlinⅡproteins were highest when the induction concentration of IPTG and lactose were0.8mmol/L and10mg/mL, the OD600of the cultured cells was0.8, and the incubating time after induction was5h at37℃. The inclusion body proteins were denatured optimally with6M guanidine hydrochloride inclusion body denature reagent after washed by2M urea inclusion body purgation buffer. The denatured recombinant mabinlin Ⅱ proteins were purified by Ni-NTA affinity chromatography. The purified denatured recombinant mabinlin Ⅱ proteins with the concentration of0.2mg/mL were refolded optimally by dialysis against urea concentration gradient refolding buffer with pH8,40mmol/L glycine concentration,300mmol/L arginine concentration and oxidation reduction potential of10:1. After the purification and refolding of recombinant mabinlin Ⅱ (MBL-BH, MBL-ABH and MBL-AXBH), the yields of dissoluble recombinant mabinlin Ⅱ proteins were respectively1.79%,3.27%and2.76%, and the purities were respectively85.4%,81.7%and83.5%. The purified recombinant proteins (MBL-BH, MBL-ABH and MBL-AXBH) expressed in E. coli could be detected by western blot using anti-mabinlin Ⅱ polyclonal antibody. Meanwhile, the purified recombinant proteins (MBL-BH, MBL-ABH and MBL-AXBH) were indentified as mabinlin Ⅱ by MALDI-TOF-TOF. The purified recombinant protein MBL-BH tasted sweetness, which was around100times sweeter than sucrose on weight basis. Moreover, the purified recombinant protein MBL-BH was not heat stablilty.3. When MBL-B and MBL-AB fused with SUMO molecular chaperone were expressed in E. coli, part of recombinant mabinlinⅡ proteins (SUMO-MBL-B and SUMO-MBL-AB) were soluble. The yields of the fused soluble protein were highest when the induction concentration of IPTG and lactose were0.4mmol/L and6mg/mL, the OD600of the cultured cells was0.4, the incubating time after induction was4h at28℃and shaking speed was150rpm. The fused recombinant proteins were purified by Ni-NTA affinity chromatography. After the purification, the yields of the soluble fused recombinant mabinlinⅡ proteins (SUMO-MBL-B and SUMO-MBL-AB) were respectively3.78%and4.23%, and the purities were respectively76.3%and75.6%. The purified fused proteins (SUMO-MBL-B and SUMO-MBL-AB) expressed in E. coli could be detected by anti-mabinlin Ⅱpolyclonal antibody immunodetection using western blot. The purified fused protein SUMO-MBL-B tasted sweet, which was around100times sweeter than sucrose on a weight basis. Moreover, the fused protein SUMO-MBL-B was heat stablilty to some extent.4. Recombinant mabinlinⅡ proteins (MBL-BH and MBL-ABH) were detected by western blot in cultured supernatants and intracellular cells when they were secretively or intracellularly expressed in food-grade L. lactis, expectively. The ELISA results showed that the recombinant mabinlinⅡ proteins were optimally expressed when the nisin induction concentration was30ng/mL, the OD600of the cultured cells was0.5, and the incubating time after induction was3h. The yields of the recombinant proteins were12.83μg/mL and15.68μg/mL when MBL-BH and MBL-ABH were secretively expressed in L. lactis, and those were22.07μg/mL and27.19μg/mL when MBL-BH and MBL-ABH were intracellularly expressed in L. lactis.In conclusion, separation and purification systems for recombinant mabinlinⅡ proteins (both inclusion bodies and fused proteins) expressed in E. coli were successfully set up for the first time; and food-grade induction expression systems for recombinant mabinlinⅡ proteins (both secretion expression and intracellular expression) in L. lactis were also successfully set up for the first time. Besides, natural active mabinlin Ⅱ protein was extracted and purified from the seeds of C. masaikai and anti-mabinlin Ⅱ polyclonal antibody with high titer was prepared using natural mabinlinⅡ as antigen. Evaluation systems of protein identification, sweet-taste activity and heat stablilty were also established for recombinant mabinlin Ⅱ proteins. Recombinant proteins MBL-BH and SUMO-MBL-B with slight sweet-taste but not heat stable were successfully acquired by E. coli expression system. Sweet-taste activity and heat stability tests implied that B-chain of mabinlin Ⅱ possessed sweet-taste activity structure but not having heat stability structure. |
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