Optimization Of Expression And Safety Evaluation Of The Multi-copy Human/Salmon Chimeric Calcitonin In Saccharomyces Cerevisiae

Calcitonin (CT) is a polypeptide of 32 amino acids, which was found to reduce bone resorption by direct inhibition the activity of osteoclast. The peptide also can promote the deposition of calcium in the bone, thereby reduce the calcium concentration of blood. Due to these bioactivities, calcitonin has been used to treat Paget’s disease of bone, osteoporosis, and hypercalcemia. Clinical studies have shown that freeze-dried Saccharomyces cerevisiae showed a high tolerance to the digestive conditions of the artificial gastrointestinal environment and calcitonin could directly go through the epidermal cells of the gastrointestinal tract into the blood in the form of peptide. As a result, our lab has constructed two different recombinant S. cerevisiae yAGA2-sCT and yAGA2-r-sCT respectively by Sun Pingnan and Jiang Yong, which made a chance for oral absorption of salmon calcitonin for human. In order to further increase the expression of calcitonin and the biological activity, a strain of S. cerevisiae YS-5hsCT expressing 5 copies of hsCT was constructed and showed the activity in the experiments in vivo.Optimization of expression and fermentation for YS-5hsCT is carried out to obtain high production of hsCT. At the same time, safety evaluation is performed according to aseries of toxicological experiments. The study will supply the experimental data for application of CT by oral absorption.The rDNA2-ura3-Ppgk-5hsCT-rDNA1 fragments is obtained with HpaⅠ digestion from plasmid pUC18-rDNA2-ura3-Ppgk-5hsCT-rDNA1 and transformed into an uracil-deficient strain of S. cerevisiae YS-△ ura and a new strain S. cerevisiae YS-5hsCT was obtained. Result of Southern blotting indicated that the human-salmon chimeric calcitonin gene was integrated in the genome of YS-5hsCT with multiple copies. Detected by Western blotting and ELISA, recombinant 5hsCT protein is expressed in the yeast. The expression level reached 2.05% of total proteins. S. cerevisiae YS-5hsCT was detected the function of decrease serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg·BW decreased serum calcium by 0.498 mM.Expression ofchimeric human/salmon calcitonin in S. cerevisiae YS-5hsCT was optimized by the analysis of transcription level of 5hsCT mRNA and protein expression level of 5hsCT under different conditions. Then single external factor experiment was performed to find the appropriate growth conditions of S. cerevisiae YS-5hsCT. At last, expression and fermentation conditions of S. cerevisiae YS-5hsCT were optimized by orthogonal test. The results showed that:(1) For expression of 5hsCT, the optimal time is 96 h, the optimal temperature is 30℃ and the optimal rotate speed is 225 rpm. (2) In single factor experiment, the optimal conditionsfor the growth of S. cerevisiae YS-5hsCT include the initial inoculation amount of 8%, the incubation time of 72 h, temperature of 30℃, rotate speed of 200-250 rpm, aeration of 7.0～8.51/min. (3) Overall consideration of the expression of 5hsCT protein and the growth of S. cerevisiae YS-5hsCT, the optimal combination is:the initial inoculation amount of 8%, incubation time of 72 h, temperature of 30℃, rotate speed of 225 rpm, and aeration of 7.01/min. Under the condition of optimum combination, the content of 5hsCT is up to 2.08% of total proteins, biomass of S. cerevisiae YS-5hsCT is reached to 7.51 g/l·d and the product of 5hsCT is up to 156.21 mg/l·d.To evaluate the toxicological safety of the recombinant S. cerevisiae YS-5hsCT, acute toxicity test, genetoxic tests (bone marrow cell erythrocyte micronueleus test, sperm shape abnormality test), traditional teratogenieity test and 30 days feeding test are performed. YS-5hsCT was considered to have no acute toxicity (LD50> 10.00 g/kg). The results of genetoxic tests showed that the micronucleus rate and sperm shape abnormality rate are not significantly different compared with the control, so YS-5hsCT has no genetoxicity, (P>0.05), The appearance, viscera, skeleton of fetal mice and reproduction of female mice are not affected by S. cerevisiae YS-5hsCT, so there are no mutagenic toxicity or embryotoxicity in teratogenic test, No significant toxicity is also detected in 30 days feeding test. The maximum tolerated oral dose of YS-5hsCT is more than 5.0 g/kg BW in mice, which is higher than the recommended dose of 0.8 g/kg BW for human. In a conclusion:the recombinant S. cerevisiae YS-5hsCT is safe and nontoxic.Our work was devoted to construct an industrial recombinant strain of S. cerevisiae YS-5hsCT without antibiotic markers for the first time. This strain was carried a multi-copy human/salmon chimeric calcitonin gene, and remained genetic stability in batches. Through the optimization of culture and expression conditions and safety evaluation, the work provides the basis for the application of oral administrated recombinant calcitonin.