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The Sweet Protein Brazzein expression in Escherichia coli and Bacillus subtilis

Author: ZhangYongChun
Tutor: ZhaoGuoPing
School: Fudan University
Course: Microbiology
Keywords: sweet protein Escherichia coli fusion expression Bacillus subtilis homologous recombination
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 58
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Abstract


Sweet protein is the natural good sweetener with low calorie.So far seven sweet proteins have been isolated and researched:Brazzein,Curculin,Mabinlin,Miraculin, Monellin,Pentadin and Thaumatin.Among those the smallest protein Brazzein was firstly isolated from wild in the fruit of west Africa plant Pentadiplandra brazzeana Baillon by D.Ming in 1994.Brazzzein is a single-chain protein containing 54 amino acids and its compact structure is supported by four intramolecular disulfide bridges. The relative molecular mass ofbrazzein is 6.5kDa and its three-dimensional structure has been solved.It is reported to be 2000 times sweeter than sucrose.Its sweetness profile is maintained even after incubation at 80℃for 4 h and 98℃for 2 h.Brazzein is different to sucrose that its absorbtion does not depend on insulin.It can replace the traditional small molecular saccharide to be the promising food additive used in healthier and natural products,but it can only get low yield with high cost by directly isolating Brazzein from plant.Although expressing Brazzein in E.coli has already come true,the amount of the target protein we get is small and this leads to the lack of protein activity.Based on the research done so far,this thesis will display the work we carried on in two prokaryotic cell expression systems and the active protein product Brazzein we got.Firstly,we compared the expression of brazzein DNA fused or not fused to E. coli signal sequence,and found that the one without signal sequence expressed in soluble form with activity.Then,we researched on the expression of brazzein gene fused with GST protein encoding sequence and bring the Xa factor cutting site in between gst and brazzein gene,and slice the fusion GST protein to release Brazzein without tag in vitro,from which we got large amount of purified no-tag Brazzein.In addition,we insert the short peptide（Gly）5 encodeing sequence in between（His）6 encoding sequence and brazzein gene for purifing if needed,and the research result demonstrated that this kind of Brazzein fusion protein dose influence the activity of Brazzein.Secondly,we for the first time try to expresse it in the food-safety host cell-Bacillus subtilis.We synthesized the Brazzein encoding sequence proper for expression in B.subtilis according to its codon preference.Then we cloned the signal sequence of amyE gene in Bacillus subtilis BF7658 genome DNA,and fused it with brazzein gene for the expression in Bacillus subtilis WB600 but we did not get the target secreted Brazzein protein.Furthermore,we brought a site mutation 31His→Ala imo the Brazzein peptide chain and synthesize this mutated gene for expression in B.subtilis WB600.After fermentation we got the soluble form of Brazzein as part of the protein products and taste it to find that it has the sweet activity.This idea is based on a report that this site mutation can make the protein twice the sweetness of the wild Brazzein.The host cell we applied in this research B.subtilis WB600 is a strain deficient in six extracellular proteases.

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