Secretion expression and activity assay of a novel fusion protein of thrombopoietin and interleukin-6 in Pichia pastoris.

Abstract

Thrombopoietin (TPO) is an important haematopoietic 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 pPICZalpha-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.