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Journal of Biochemistry Advance Access published online on May 21, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm111
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© 2007 The Japanese Biochemical Society

Secretion Expression and Activity Assay of a Novel Fusion Protein of Thrombopoietin and Interleukin-6 in Pichia pastoris*

Hu Zhiyi1, Lai Wenshan2, Zhang Wenze3, Deng Ning4, Zhang Chi2, Ye Kenan3, Wang Ping3, Wang Qianqian3 and Zhang Qing3,**

1State Key Discipline Department Of Internal Medicine, The First School Of Medicine ,Guangzhou University of Chinese Medicine 510405, China; 2School of Pharmaceutical Sciences, Zhongshan(SUN YAT-SUN) University, Guangzhou, 510275, China; 3Biotechnology Research Center, Key Laboratory Of Gene Engineering of the Education Ministry, School of Life Science, Zhongshan(SUN YAT-SUN) University, Guangzhou, 510275, China; 4College of Life Science and Technology, Jinan University, Guangzhou, 510632, China.

** Corresponding author: Zhang Qing. Biotechnology Research Center, Key Laboratory Of Gene Engineering of the National Education Ministry, School of Life Science, Zhongshan(SUN YAT-SUN) University, Guangzhou, 510275, China. Tel: 86-20-84113988, E-mail: lsszq{at}mail.sysu.edu.cn

Received March 4, 2007; Accepted March 22, 2007


  Abstract

Thrombopoietin (TPO) is an important hematopoietic factor in megakaryocytic activities as well as in platelet production. Interleukin 6 (IL-6) can co-stimulate TPO-dependent formation of colony forming unit of megakaryocyte (CFU-Meg) growth which could be responsible for residual platelet formation in TPO-deficient or c-mpl-deficient animals. In this report, we demonstrated the development of a high-level expression system to produce a 78 kDa human fusion protein IL-6/TPO (named ZH646). This was achieved by constructing the expression vector pPICZ{alpha}-A-IL-6-linker-TPO, and obtained the recombinant yeast GS115, which then efficiently secreted into a medium with a yield of 30 mg/L from the supernatant of the yeast culture in flask. ZH646 was then purified using two steps via DEAE-Sephacel chromatography and Mono Q columns. Activity assay showed that ZH646 could significantly stimulate the formation of CFU-Meg and the proliferation of Dami cells in vitro in a dose-dependent manner. In addition, ZH646 also showed thrombopoietic effect in normal mice, and the ability to enhance recovery of normal platelet counts after myelosuppression mice. These results suggested that ZH646 is a novel protein, and its activities are much stronger than that of TPO or IL-6 alone. ZH646 therefore has a broad spectrum of megakaryopoiesis activity associated with platelet production.

Key Words: fusion protein (IL-6/TPO), expression, purification, platelet production, Pichia pastoris

*This work was supported by grants from the National Natural Science Foundation Of China(4103250 and 30572453), the National Education Ministry Key Science and Technology Research Project (199177), Guangdong Natural Science Foundation (4203208) and Guangdong Provincial Social Development Plan (2005B20301024).


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