Expression Of Sweet Protein Monellin In Escherichia Coli

According to the monellin amino acid sequence and Escherichia coli preference codon usage, the monellin gene was designed and synthesized. Then it was cloned into vector pET22b and pET28a, and the recombined plasmid pET22b-Mon and pET28a-Mon were transformed into E. coli BL21(DE3). The recombinant might improve monellin stability upon temperature or pH changes. Lactose was used as an inducer instead of IPTG, which might reduce the production cost. And compared the influences of two different vectors(pET22b/pET28a) on the expression of Monellin.The synthesized monellin gene was cloned in the pET22b vector by using NcoⅠ/BamHⅠcutting sites. Recombinant plasmid pET22b-Mon was gotten, which was then transformed into E. coli BL21(DE3) to obtain the recombinant strain- DE3-pET22b-Mon.The monellin gene was cut from pET22b-Mon by NcoⅠ/BamHⅠ, and was ligated in pET28a. Recombinant plasmid pET28a-Mon was gotten, which was then transformed into E. coli BL21(DE3) to obtain the recombinant strain- DE3-pET28a-Mon.The induction conditions on the expression of Monellin were optimized when inducing by IPTG. As for DE3-pET22b-Mon, it was grown in 100 mL shake flask with 20mL LB at 37℃. Vaccination quantity was 1%. When the culture OD600nm reached 0.73, IPTG was added to give a final concentration of 1mmol/L. The culture was then incubated at 37℃for 6h and the Monellin content reach 22.26% of total proteins. As for DE3-pET28a-Mon, it was grown in 100mL shake flask with 20mL LB at 37℃. Vaccination quantity was 1%. When the culture OD600nm reached 1.0, IPTG was added to give a final concentration of 1mmol/L. The culture was then incubated at 37℃for 6h and the Monellin content reach 34.08% of total proteins.The induction conditions on the expression of Monellin were optimized when inducing by lactose. As for DE3-pET22b-Mon, it was grown in 100mL shake flask with 20mL LB at 37℃. Vaccination quantity was 3%. When the culture OD600nm reached 1.0, lactose was added to give a final concentration of 10g/L. The culture was then incubated at 37℃for 7h and the Monellin content reach 19.86% of total proteins. As for DE3-pET28a-Mon, it was grown in 100mL shake flask with 20mL LB at 37℃. Vaccination quantity was 3%. When the culture OD600nm reached 1.0, lactose was added to give a final concentration of 11g/L. The culture was then incubated at 37℃for 8h and the Monellin content reach 33.09% of total proteins.To exploite feasibility of producing Monellin in large scale, we chose DE3-pET28a-Mon and lactose for fermention. The germ with OD600nm=0.8 was poured into the pilot scale fermentor(working volume is 5L), and cultured for 21h to give a final optical density of 6.02 at 600nm, the purified expressed protein yield reached 0.06g. And the newly designed protein is the same sweet as the nature one. It is more stable upon temperature or pH changes, and retains sweetness after heating to 70℃for 5min, even heating to 50℃at low pH(pH=3).