Abstract: There are 4 types of desmodus salivary plasminogen activators（DSPAs）,i.e.,DSPAα1,DSPAα2,DSPAβ and DSPAγ. DSPAα1 and DSPAα2 contain a finger domain（F）,an epidermal growth factor domain（E）,a kringle domain（K）and a serine proteinase domain（P）;DSPAβ contains E,K and P domains;and DSPAγ contains K and P. The effect of DSPAα1’s structure on fibrinolytic activity was also investigated. A deletion mutant of DSPAα1（mDSPAα1）lacking the E domain was synthesized by splicing overlap extension PCR（SOE-PCR）method,and the recombinant plasmids of mDSPAα1/pPIC9K,DSPAβ/pPIC9K and DSPAγ/pPIC9K were constructed and transformed into Pichia pastoris GS115. Fibrinolytic activity was determined by fibrin plate assay. Results showed that the expression of DSPAγ was not detected,the activities of DSPAα1,DSPAβ,and mDSPAα1 were 2.64 × 10⁵ U/mg,1.32 × 10⁴ U/mg and 151.52 U/mg respectively. The total activity hardly changed when using DSPAα1,mDSPAα1 and DSPAβ together,but there led a 2-3folds of reduction by using either two of them. The mDSPAα1 exhibited almost no fibrinolytic activity,whilst DSPAβ retained comparable catalytic activity to the wild-type DSPAα1. Deletion mutant studies illustrated that N-terminal region of DSPAα1 greatly affected the PA activity,and it without E domain nearly lost its activity,indicating that the E domain of DSPAα1 plays a key role during fibrinolytic process.

Key words: DSPAα1, SOE-PCR, deletion mutants, Pichia pastoris, fibrin plate assay