Construction and expression of recombinant staphylokinase-hirudin fusion protein

doi:10.3969/j.issn.2095-4344.2015.20.020

Abstract

Abstract:

BACKGROUND: Recombinant fusion protein is cascaded by staphylokinase and hirudin according to the thrombin cognition sequence, and has double functions and a molecular weight of 23 ku. The recombinant fusion protein can be highly expressed in the engineering bacteria at high-density fermentation.
OBJECTIVE: To construct and purify recombinant staphylokinase-hirudin fusion protein in the engineering bacteria after high-density fermentation, and to explore the feasibility of construction and the expression value.
METHODS: The engineering bacteria were cultured at high density and staphylokinase-hirudin fusion protein was induced to express. The bacteria were centrifuged and ultrafiltrated after repeated freezing and thawing. The supernatant was collected with ion exchange chromatography method. The staphylokinase-hirudin fusion protein was isolated and purified, then the fibrinolytic activity and expression in bacteria were observed.
RESULTS AND CONCLUSION: The engineering bacteria were cultured and the fusion protein was induced at 17 hours. The results showed that, staphylokinase-hirudin fusion protein expression was detected at 0.5 hours after induction, and the expression levels were increased as the fermentation time; at 20 hours, the expression level reached the peak. The dried weight of the bacteria was 32.20 g/L and the expression level of target proteins was 1.48 g/L. After purification, the purity of recombinant staphylokinase-hirudin fusion protein was as high as 98%, fibrinolytic activity was about 2.6×104 IU/mg, the probability of activity recovery was 56%. The purification process of recombinant staphylokinase-hirudin fusion protein is convenient, less time, repeatable and allows large-scale production.

Key words: Hirudins, Recombinant Fusion Proteins, Fermentation

CLC Number:

R318

Xu Hua, Dong Yun, Yang Jin, Liu Shao-bang, Liu Shao-hua, Chen Wu. Construction and expression of recombinant staphylokinase-hirudin fusion protein [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(20): 3221-3225.

Figures/Tables 1

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