Construction of Pichia pastoris strain expressing salivary plasminogen activator from vampire bat (Desmodus rotundus).
- Author:
Yan LIU
1
;
Chang SU
;
Xiaoshuang SONG
;
Yalan TANG
;
Zhenhong BAO
Author Information
1. School of Life Science, Southwest University, Chongqing 400715, China. liuyan@swu.edu.cn
- Publication Type:Journal Article
- MeSH:
Animals;
Base Sequence;
Chiroptera;
genetics;
Genetic Vectors;
genetics;
Molecular Sequence Data;
Pichia;
genetics;
metabolism;
Plasminogen Activators;
biosynthesis;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
Recombination, Genetic;
Sequence Analysis, DNA
- From:
Chinese Journal of Biotechnology
2009;25(4):566-574
- CountryChina
- Language:Chinese
-
Abstract:
Vampire bat saliva contains a plasminogen activator that presumably assists these hematophagous animals during feeding. Bat-PA (H), the full-length form of Vampire Bat Salivary Plasminogen Activator (DSPAalpha1), is homologous and similar efficacy to tissue-type plasminogen activator (t-PA). The strict fibrin dependence of activity is a characteristic which could be desirable in the fibrinolytic therapy. It is a unique fibrinolytic enzyme that does not promote neurodegeneration. In this study, according to the reported gene sequence (GenBank Accession No. J05082) of Vampire bat (D. rotundus) plasminogen activator. It was the first time to synthesize the full sequence of DSPAalpha1 in vitro and clone it into the expression vector pPIC9K, the recombinant plasmid was linearized and transformed into Pichia pastoris GS115 strain. Secreted expression of recombinant DSPAalpha1 was attained by methanol induction and its molecular mass is 47 kD. To get recombinant GS115 with high amount of protein, hundreds of His+ transformants had been screened to isolate clones resistant to high levels G418 (2-4 mg/mL), the selected clones mini-expressed in Pichia pastoris, and tested their fibrinolytic activities and expressed protein bands by fibrin plate assay and SDS-PAGE. DSPAalpha1 was determined by optical density after SDS-PAGE, the yield is about 30 mg per liter of fermentation culture. DSPAalpha1 derived often from mammalian cells: Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, COS cells, which might be produced at high cost. In Pichia pastoris, it is expected to higher yield and lower cost, thus it might be able to serve as new thrombolytic candidate.
