Abstract
Objectives
To develop a novel thrombopoietin (TPO) analog by fusing the tandem TPO mimetic peptide (TMP-TMP) to human serum albumin (HSA) and performing functional expression of recombinant fusion protein HSA-TMP-TMP.
Results
After optimizing the fusion orientation in shake-flask culture, HSA-TMP-TMP was expressed at 0.4 g/l in Pichia pastoris grown in a 20 l bioreactor, during which pH was controlled at 5 by addition of NH4OH and citric acid. The fusion protein significantly activated signal transducer and activator of transcription-mediated transcription in TPO receptor-dependent manner, which was demonstrated by a luciferase reporter assay. Following subcutaneous administration, HSA-TMP-TMP effectively stimulated the platelet production in healthy mice in a dose-dependent manner.
Conclusion
Successful expression of HSA-TMP-TMP fusion protein in P. pastoris was achieved and the recombinant HSA-TMP-TMP is a promising TPO analog.
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Acknowledgments
This study was supported by the Special International Cooperation Project of Ministry of Science and Technology (2012DFA30480), The Key Project of Gansu Province Science and Technology (1002WKDE055), Gansu Province Scientific Research Project Fund (090WCGA900), Fundamental Research Funds for the Central Universities of China (lzujbky-2014-147) and Science and Technology Project of Lanzhou (2010-1-154).
Supporting information
Supplementary Table 1—List of primers used in construction of pPinkα-TMP-TMP-HSA and pPinkα-HSA-TMP-TMP.
Supplementary Fig. 1—Construction and schematic representation of pPinkα-TMP-TMP-HSA and pPinkα-HSA-TMP-TMP expression plasmids. The nucleotide sequence encoding TMP-TMP-HSA and HSA-TMP-TMP were amplified by two-step overlap extension PCR and inserted into pPinkα-Hc by infusion technology, respectively.
Supplementary Fig. 2—Optimization of culture pH for HSA-TMP-TMP expression in shake flasks. Protein expression in shake flasks at indicated pH were examined by reduced SDS-PAGE. Nine pH conditions were tested, in which pH 5 showed the highest expression level. Line M represents the protein molecular weight marker.
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Wang, M., Zhi, D., Xian, J. et al. Functional expression of human serum albumin-tandem thrombopoietin mimetic peptide fusion protein as a novel thrombopoietin analog in Pichia pastoris . Biotechnol Lett 38, 779–785 (2016). https://doi.org/10.1007/s10529-016-2049-8
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DOI: https://doi.org/10.1007/s10529-016-2049-8