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Huang Y.-C.,University of Sichuan | Gou X.-H.,Pharmaceutical Institute Of Chengdu Of Ao Pharmaceutical Group | Han L.,Pharmaceutical Institute Of Chengdu Of Ao Pharmaceutical Group | Li D.-H.,Pharmaceutical Institute Of Chengdu Of Ao Pharmaceutical Group | And 2 more authors.
Journal of Sichuan University (Medical Science Edition) | Year: 2011

Objective To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. Methods The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS) 3 by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). Results Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 IU/(mL · 10 6 · 72 h) to 637 IU/(mL · 10 6 · 72 h). Conclusion The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.

Huang Y.,University of Sichuan | Gou X.,Pharmaceutical Institute Of Chengdu Of Ao Pharmaceutical Group | Gou X.,Chengdu University of Technology | Hu H.,Pharmaceutical Institute Of Chengdu Of Ao Pharmaceutical Group | And 3 more authors.
Journal of Applied Microbiology | Year: 2012

Aims: S-adenosyl-l-methionine (SAM) is an important biochemical molecule with great potential in the pharmacological and chemotherapeutic fields. In this study, our aims were to enhance SAM production in Saccharomyces cerevisiae. Methods and Results: Through spaceflight culture, a SAM-accumulating strain, S. cerevisiae H5M147, was isolated and found to produce 86·89% more SAM than its ground control strain H5. Amplified fragment length polymorphism (AFLP) analysis demonstrated that there were genetic variations between strain H5M147 and its ground control. Through recombinant DNA technology, the heterologous gene encoding methionine adenosyltransferase was integrated into the genome of strain H5M147. The recombinant strain H5MR83 was selected because its SAM production was increased by 42·98% when compared to strain H5M147. Furthermore, cultivation conditions were optimized using the one-factor-at-a-time and Taguchi methods. Under optimal conditions, strain H5MR83 yielded 7·76gl -1 of SAM in shake flask, an increase of 536·07% when compared to the strain H5. Furthermore, 9·64gl -1 of SAM was produced in fermenter cultivation. Conclusions: A new SAM-accumulating strain, S. cerevisiae H5MR83, was obtained through spaceflight culture and genetic modification. Under optimal conditions, SAM production was increased to a relative high level in our study. Significance and Impact of the Study: Through comprehensive application of multiple methods including spaceflight culture, genetic modification and optimizing cultivation, the yield of SAM could be increased by 6·4 times compared to that in the control strain H5. The obtained S. cerevisiae H5MR83 produced 7·76gl -1 of SAM in the flask cultures, a significant improvement on previously reported results. The SAM production period with S. cerevisiae H5MR83 was 84h, which is shorter than previously reported results. Saccharomyces cerevisiae H5MR83 has considerable potential for use in industrial applications. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

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