Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites

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• 作者：Hengyi Sun ; Xiaonan Zang ; Yuantao Liu…
• 关键词：Chimeric human/salmon calcitonin ; Expression ; Multi ; copy integration ; rDNA ; Saccharomyces cerevisiae
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：99
• 期：23
• 页码：10097-10106
• 全文大小：1,251 KB
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• 作者单位：Hengyi Sun (1)
  Xiaonan Zang (1)
  Yuantao Liu (1)
  Xiaofei Cao (1)
  Fei Wu (1)
  Xiaoyun Huang (1)
  Minjie Jiang (1)
  Xuecheng Zhang (1)
  
  1. Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, 5 Yushan Road, Qingdao, 266003, People’s Republic of China
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

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.