Turn Salmon Calcitonin Gene Yeast Optimization Of Fermentation Conditions And Safety Evaluation

Calcitonin (CT), a polypeptide hormone consisting of 32 amino acids with an N-terminal disulfide bridge and a C-terminal prolineamide residue. It is origined from the thyroid C cells in mammals and secreted from ultimobranchial body in fish. Four forms of CT are used clinically, namely synthetic human CT (hCT), synthetic salmon CT (sCT), natural porcine CT (pCT), and a synthetic analogue of eel CT. They are widely utilized as bone resorption inhibitors for the treatment of osteoporosis, hypercalcemia, Paget's disease, and rheumatoid arthritis. Recombinant saccharomyces cerevisiae expressing salmon calcitonin, namely yAGA3-r-sCT, is showed the activity in the experiments in vivo by Jiang Yong and it makes a chance for oral absorption delivery system of calcitonin. Optimization research of fermentation for yAGA3-r-sCT is carried out to obtain high production and safety evaluation is necessary which is performed by a series of toxicological experiments. The results will supply the experimental data for application of CT by oral absorption..Optimization of fermentation for yAGA2-r-sCT includes several parts as follows. Orthogonal design is used to improve primary medium composition for production used in the lab, and the optimal medium composition is obtained as follws:6% defatted soybean hydrolysate (DSH),3%glucose,0.5%yeast extract,1%peptone, pH 5. One variable at a time design is used to optimize environment factors effecting on growth of yAGA2-r-sCT, and the optimal condition is obtained as follows: temperature 28℃, agitation 200 rpm, aeration 8 L/min, Inoculum volume8%, fermentation time 3 days. Fractional factorial design is used in the first step to evaluate the effects of 6 variables on industrial medium composition and DSH, glucose, NaCl are screened as significant factors. Then, their response region close to the maximum production is obtained by steepest ascent design. Their concentrations are further optimized using response surface methodology based on central composite design. The optimum predicted medium for maximum production of recombinant sacchromyces cerevisiae yAGA2-r-sCT is found to comprise:86.5 g/L DSH,56.8 g/L glucose,1.10 g/L NaCl,5.0 g/L K2HPO4,0.6 g/L (NH4)2SO4,0.2 g/L MgSO4. At the end, Optimization of fermentation for inducing expression of sCT is found to comprise:initial OD600 0.8, induction time 12 h, galactose 2% added at one time. The maximum expression of sCT is 5.92%.To evaluate the toxicological safety of recombinant sacchromyces cerevisiae yAGA2-r-sCT, tests include acute toxicity test, genetoxic tests (bone marrow cell erythrocyte micronucleus test, sperm shape abnormality test), traditional teratogenicity test,30-days feeding test, and long-term toxicity test for 90 days are performed. Results:yAGA2-r-sCT has no acute toxicity (LD50>10.0 g/kg).The results of genetoxic tests shows that the micronucleus rate and sperm shape abnormality rate are not significantly different compared with the control, so yAGA2-r-sCT has no genetoxicity or nutation. (P>0.05). The appearance, viscera, skeleton of fetal mice and reproduction of female mice are not affected by yAGA3-r-sCT, so there are no maternal toxicity or embryotoxicity in teratogeniccity test. No significant toxicity is detected in 30 days feeding test and long-term toxicity test for 90 days. The maximum tolerated oral dose of yAGA2-r-sCT is more than 5.0 g/kg-BW in mice while human recommended dose is 0.8 g/kg·BW. Conclusion: recombinant Saccharomyces cerevisiae expressing salmon calcitonin is safe and nontoxic.