Cloning And Expressing Of Brazzein Gene

There are seven known sweet-tasting proteins: Thaumatin,Monellin,Mabinlin, Miraculin,Pentadin, Curculin and Brazzein.They have advantageousproperties of being nontoxic, low caloric and noncariogenic. So they have theprobability to take place of sugar. These proteins can elicit sweet taste thoughtheir structure are different from each other. Brazzein originally separated from the fruit of the African plantPentadiplandra brazzeana Baillon. Brazzein is a 6.5 kDa protein with fourdisulfide bonds. Brazzein has been sequenced, it is a single chain protein. It is thesmallest, most heatstable and pH stable sweet protein known to date. There aretwo forms of Brazzein: the major form(54 amino acids) and the minor form(53amino acids). The two forms are identical except for absence of a pyroglutamicacid at the amino terminus of the minor form. Brazzein genes were transformed in to E.coli to study the structure activityrelationship. But there are no further exact reports about the applied study ofBrazzein. In order to get sufficient quantities of pure biologically activeBrazzein to meet the need of basic research and clinical use, we cloned the geneof Brazzein into pET plasmids, a series of prokaryotic expression vector, andexpressed the recombinant plasmids in the E.coli of BL21(DE3). 1. Brazzein gene were cloned by way of PCR, then ligated the genes ofBrazzein to the vector of T-easy, the results of sequencing indicate that thesequences of Brazzein are correct. 2.Brazzein gene were achieved by PCR using a pair of oligo-nucleotideprimers to introduce to the suitable restriction enzyme site, and the Brazzeinproduct of PCR contains 180 base pairs. 3. The gene of Brazzein were ligated into the expression vector pET-32a (+)and pET-30a(+) after cut by the restriction enzymes. We transfered therecombinant vector pET-32a(+)-Brazzein and pET-30a(+)-Brazzein into the E.coli of BL21(DE3) after encoding fragment of recombinant vectors constructcorrectly that confirmed by sequencing. 4. SDS-PAGE analysis suggested that the bacteria containing therecombinant plasmid pET-32a (+)-Brazzein produced the fusion protein ofabout 26kDa as it was induced by IPTG, consisting 20% of the total bacterialproteins. The recombinant plasmid pET-30a (+)-Brazzein produced the fusionprotein of about 12kDa as it was induced by IPTG, consisting 25% of the totalbacterial proteins. 5. We find that the fusion proteins are inclusion bodies when the bacteriaare lysised by the lysozyme.