| Today, the role of carbohydrates in the food industry is very important. It is one of the major flavor additives that currently used, and the lack will seriously affect food flavor. However, there are several problems associated with their long-term and numerous intakes, such as high caloric content, tooth decay, diabetes mellitus and so on, they are a tremendous threat to human health. This leads to demand for non-sugar alternatives can be used in foods, beverages, and medicines, but most non-sugar sweeteners either have unwanted side effects or temporal sweetness profiles that do not adequately match that of sugar. The discovery of sweet proteins opens a whole new chapter to solve this problem.Sweet proteins are the proteins that can evoke a sensation of sweetness. Since 1970s, there are seven sweet proteins intermittently derived from the fruits of tropical plants, such as miraculin, monellin, mabinlin, haumatin, curculin, brazzein and pentadin. No doubt, they are very good sweet additive. However, no common features have been observed among these proteins in amino acid sequence or in tertiary structure. Brazzein was isolated from the fruit of Pentadiplandra brazzeana Baillon. It is the smallest and one of the sweetest of the protein sweeteners discovered so far, and combines a long history of human consumption, small size with high sweet potency, solubility and exceptional thermo stability. It is composed of a single chain of 54 amino acid residues, molecular weight is 6.5 KD and isoelectric point is 5. The molecule is 3,500 times sweeter than sucrose on a molar basis and 2,000 times sweeter than sucrose on a weight basis. It tastes purely sweet with no sourness, saltiness or bitterness. It is exceptionally heat stable and its sweet taste remains after incubation at 98°C for 2 h and at 80°C for 4.5 h in the pH range of 2.5–8, this may be the result of its four intramolecular disulfide bonds and lack of free sulfhydryl groups. It often improves the mouth feel of beverages when blended with other sweeteners and works well in both citric acid and phosphate beverage systems.In recent years, there has been an increasing demand for healthy and natural sweeteners. Therefore, and in order to address this need, there is an intense and ongoing search for methods of production. But extraction of Brazzein from the natural source can become a complex problem, as the producing plants appear to have difficulties in bearing fruit outside the natural habitats. Depending on the market need for the particular product, extraction from the natural plant might not be able to keep up with demand. Chemical synthesis is uneconomical basing on the cost consideration. So we attempt to express the sweet protein Brazzein in a recombinant host. However, the broad application of prokaryotic expression system with proteins derived from eukaryotic genomes that require post-translational modification(s) has been problematic because this simple microorganism lacks the intracellular machinery to achieve these outcomes. So we choose eukaryotic expression system to product Brazzein. Methylotrophic yeast Pichia pastoris and mammalian cells are the major host for complex recombinant protein expression. They are suitable for use in expression studies with proteins that contain a high level of disulphide connectivity or proteins that require other types of posttranslational modifications such as glycosylation, proline cis/trans isomerization, disulphide isomerization, lipidation, sulphation or phosphorylation.In this paper, according to the amino acid sequences of the sweet protein Brazzein, the biased codons of Pichia pastoris and the latest research advances about the protein's structure and mechanism, we reformed the gene of Brazzein, which includes 3 sites of mutation(Asp29Lys, His31Ala, Glu41Lys). The Brazzein gene was amplified by SOE-PCR, then sequenced.The Brazzein gene which has the right sequence was connected to plasmid pGAPZαA, So we get the recombinant plasmid pGAPZαA-Bra. The pGAPZαA-Bra was linearized by digestion with AvrⅡ. Competent P. pastoris SMD1168 was transformed by electroporation using electroporater. The ZeocinTM positive strains was screened, then cultured in YPD medium at 25℃. Cultured for 3 days, Culture supernatants concentrated by vacuum freeze drying were analyzed with SDS-PAGE in 15% gel, the comeout agreed with expected results. Culture supernatants were purified by Ni-ion affinity chromatography. The purified fusion protein was cleaved by thrombin. Sensory analyses show that the Brazzein protein containing mutation is 150 times sweeter than a sucrose solution on a weight basis, meanwhile, the protein without mutation is 100 times sweeter. The supernatants of culture, at various time interval(12h, 24h, 48h, 72h, 96h)and temperature gradient(20℃, 23℃, 25℃, 28℃, 30℃), were collected, then were concentrated and purified. The maximum expression of Brazzein was 9.2 mg/L when cultured at 23℃for 72h. The Brazzein gene was inserted into plasmid pcDNA3.1, then transferred into mammalian cell COS-7 cells with Lipofectamine 2000. After transfection, total cellular RNA was extracted using Trizol in accordance with the manufacturer's instructions when cells are cultured for 2 days and performed reverse transcription. Brazzein expression at the transcription level was detected by Real-Time PCR, meanwhile, GAPDH transcripts were evaluated in this study. The number of copies of Brazzein mRNA is 7753, it is 18 times than GAPDH transcripts. Due to Brazzein molecule was expressed at very lower level from cells, expression at the protein level was not detected.In our opinion, Brazzein molecule has been expressed using Pichia pastoris and mammalian cells expression systems. These two expression systems can be further optimized so that more products can be obtained.
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