caa a22 4500 605504636 CHVBK 20210128100617.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6834-4 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6834-4 Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites [Elektronische Daten] [Hengyi Sun, Xiaonan Zang, Yuantao Liu, Xiaofei Cao, Fei Wu, Xiaoyun Huang, Minjie Jiang, Xuecheng Zhang] Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-P pgk -5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1kb (rDNA1) and 1.4kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-P pgk -5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-P pgk -5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04% of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis. Springer-Verlag Berlin Heidelberg, 2015 Chimeric human/salmon calcitonin nationallicence Expression nationallicence Multi-copy integration nationallicence rDNA nationallicence Saccharomyces cerevisiae nationallicence Sun Hengyi Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut Zang Xiaonan Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut Liu Yuantao Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut Cao Xiaofei Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut Wu Fei Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut Huang Xiaoyun Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut Jiang Minjie Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut Zhang Xuecheng Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10097-10106 0175-7598 99:23<10097 2015 99 253 https://doi.org/10.1007/s00253-015-6834-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6834-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sun Hengyi Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut NATIONALLICENCE 700 1- Zang Xiaonan Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut NATIONALLICENCE 700 1- Liu Yuantao Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut NATIONALLICENCE 700 1- Cao Xiaofei Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut NATIONALLICENCE 700 1- Wu Fei Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut NATIONALLICENCE 700 1- Huang Xiaoyun Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut NATIONALLICENCE 700 1- Jiang Minjie Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut NATIONALLICENCE 700 1- Zhang Xuecheng Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, 266003, Qingdao, People's Republic of China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10097-10106 0175-7598 99:23<10097 2015 99 253