Preliminary Research On The Expression Of Thaumatin Gene In Saccharomyces Cerevisiae

Thaumatin is a sweet-tasting protein isolated from the arils of Thaumatococcusdaniellii Benth, a plant native to tropical West Africa in1855. With a sweetnessthreshold of50nmol/L in humans, thaumatin is one of the world’s sweetest substancesaccording to the Guinness Book of World Records. Maturate thaumatin consists of asingle-chain protein of207amino acid, with an isoelectric point of12and molecularmass of22kDa. At the isoelectric point, thaumatin has six intensely sweet forms, withtwo major components (thaumatin I and II). Thaumatin contains eight intramoleculardisulphide bonds(9～204，56～66，71～77，121～193，126～177，134～145，149～158，159～164). The disulfide bonds are important for maintaining and restorecorrect conformation.The habitat of Thaumatococcus daniellii Benth is very strict. In these foreignhabitats (including greenhouses), the plant grows but bears no fruit. So the difficultyin and the limitation of obtaining the natural source of thaumatin. Production ofthaumatin using genetic engineering may be an alternative to the extraction of thisproduct from fruits. The sweet-taste of recombinant thaumatin is dependent on propercorrect conformation. In addition, as a sweetener, the production of thaumatin must becorresponded with food products. So we express a large quantity of thaumatin in theSaccharomyces Cerevisiae expression system for sensory analysis of sweet protein.Saccharomyces Cerevisiae have the ability to perform higher eukaryotic proteinmodifications, such as glycosylation, disulphide bond formation and proper folding,which is important for the sweetness of thaumatin.In this study, we designed the nucleotide acid sequences of thaumatin Ⅰaccording to amino acid sequences of thaumatin Ⅰ and Saccharomyces Cerevisiae codon preference. A restriction enzyme site EcoR I is divided thaumatin gene into thefragment of A and B. Connected through EcoR I, the successful sequencing A and Bfragments are linked to Saccharomyces Cerevisiae codon preferred thaumatin codingsequences. For getting tha his-tag gene, we design6×his-tag primer and use thesuccessful sequencing of tha gene as template for PCR. Tha and tha his-tag gene weresubcloned onto the pYES2expression vector. By LiAC transformation, pYES2-Tha，pYES2-Tha his-tag and pYES2were transformed into Saccharomyces CerevisiaeINVSc1. PCR assays determined the clones on SC-U for the gene target. To induceexpression of thaumatin from the GAL1promoter,2%galactose is added to themedium. Harvest cells at0,24,48and72hours after galactose induction. Afterpreparing a cell lysate from positive transformants by acid-washed glass beads, about70%cells were broken. We can analyze the supernatant of cell lysate by SDS-PAGEand western blot. Compare with negative control, pYES2-Tha and pYES2-Tha his-tagtransformants haven’t a22kDa band after induction. We presume unreasonableinducing conditions affect the level expression of thaumatin in Saccharomycescerevisiae.The GFP gene was digested from recombinant plasmid pMD18T-GFP, and wassubcloned onto the pYES2expression vector. We observed GFP fluorescence underfluorescent microscopic, and found four characteristics:(1) Under the same inducingcondition, with extend induction time, the number of yeast expressing cells was fewer.(2) Compare wit different induction groups at the same induction time, we found10%galactose induced expression GFP was the most efficient.(3) Glycerol, ethanol, andIPTG (galactose analogue) had no the role of induction on GAL1promoter.(4)Glycerol can help the induction of galactose on GAL1promoter.Taking into account the expensive cost of galactose, we select the optimalinduction conditions for2%glycerol+0.5%galactose. Unfortunately, regardless ofGFP or thaumatin, they haven’t significantly induced expression bands on SDS-PAGEunder optimal induction conditions. Presumably, trace protein expression and proteindegradation lead to no expression bands on SDS-PAGE. In conclusion, we have successfully constructed Saccharomyces cerevisiaepreference Tha gene, and subcloned into yeast expression vector pYES2. Inpreliminary exploration, we chose transformants containing pYES2-Tha to operate,and made preparations for the following study. In inducible expression, we establishan efficient induction method by using the recombinant plasmid pYES2-GFP.