title: Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites.
abstract: 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 1.1 kb (rDNA1) and 1.4 kb (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 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, Qingdao, 266003, People's Republic of China.
authors.forename: Hengyi
authors.initials: H
authors.lastname: Sun
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, Qingdao, 266003, People's Republic of China. xnzang@ouc.edu.cn.
authors.forename: Xiaonan
authors.initials: X
authors.lastname: Zang
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, Qingdao, 266003, People's Republic of China.
authors.forename: Yuantao
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, Qingdao, 266003, People's Republic of China.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Cao
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, Qingdao, 266003, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wu
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, Qingdao, 266003, People's Republic of China.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Huang
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, Qingdao, 266003, People's Republic of China.
authors.forename: Minjie
authors.initials: M
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, Qingdao, 266003, People's Republic of China.
authors.forename: Xuecheng
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000284:Administration, Oral; D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D002116:Calcitonin / Q000096:biosynthesis* / Q000235:genetics*; D002118:Calcium / Q000032:analysis; D015825:Chromosomes, Fungal; D004275:DNA, Ribosomal / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D059765:Homologous Recombination; D006801:Humans; D051379:Mice; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012474:Salmon; D044967:Serum / Q000737:chemistry
chemical_list: D004275:DNA, Ribosomal; D011993:Recombinant Fusion Proteins; D002116:Calcitonin; D002118:Calcium
keywords: Chimeric human/salmon calcitonin; Expression; Multi-copy integration; Saccharomyces cerevisiae; rDNA