The thrombolytic agent DSPAalpha1 is currently undergoing clinical trials for the treatment of acute ischemic stroke and has shown good pharmacodynamic, pharmacokinetic and safety profiles. Here, the DSPAalpha1 gene, optimized for the preferred codons of yeast, was cloned into the Pichia pastoris strains GS115 and KM71. Both expression systems produced functional DSPAalpha1 into the broth medium under shaking flask growth conditions with the yield of about 70 mg/L, and 105 mg/L, respectively. In addition, three glycosylation minus DSPAalpha1 mutants, constructed by site-directed mutagenesis, were also expressed in Pichia pastoris. The mutant proteins were assayed by SDS-PAGE and fibrin degradation activities were evaluated. The secretion levels of all the mutants, especially N362Q and N117Q/N362Q, were so lower compared to the wild-type DSPAalpha1 that only minimal quantities of mutant protein could be recovered by purification from the culture medium. The protein specific activities from mutants (N117Q, N362Q) were less 25% than that of the wild type protein. These results imply that the N-linked carbohydrate chains (at N117 and N362) are vital for the enzymatic activity of rDSPAalpha1 and for its secretion from Pichia pastoris.
Fibrinolytic Agents ; metabolism ; Genetic Vectors ; genetics ; Glycosylation ; Pichia ; genetics ; metabolism ; Plasminogen Activators ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology

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.
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

The plasminogen and plasmin system, which is mainly regulated by urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1), is generally believed to play a role in cancer invasion and metastasis. This study was conducted to investigate the role of uPA, uPAR and PAI-1 in the invasion and metastasis of gastric adenocarcinoma. The expression of mRNAs for uPA and PAI-1 was determined by Northern blot analysis in nine primary gastric cancer tissues, nine paired metastatic lymph nodes and normal gastric mucosa. The mRNA of uPA was not or faintly detected in normal mucosa, while the expression was increased in both primary gastric cancer tissues and metastatic lymph nodes to a similar degree. The mRNA expression for PAI-1 in the gastric cancer tissues was not different from that in the paired metastatic lymph nodes and normal mucosae. uPAR was determined by immunohistochemical staining, demonstrating that five (56%) and six (67%) out of nine primary gastric cancer tissues and nine paired metastatic lymph nodes were positive, respectively and the intensity was stronger in metastatic lymph nodes. The results support the concept that most gastric cancer cells may have an innately moderate level of uPA and uPAR, and that increase of uPAR expression can be considered to be closely associated with cancer invasion and metastasis.
Adenocarcinoma/*metabolism/pathology ; Gastric Mucosa/metabolism ; Gene Expression ; Human ; Immunoenzyme Techniques ; Lymph Nodes/metabolism/pathology ; Neoplasm Metastasis ; Plasminogen Activator Inhibitor 1/*biosynthesis/genetics ; Plasminogen Activators/*biosynthesis/genetics ; RNA, Messenger/biosynthesis ; Receptors, Cell Surface/*biosynthesis/genetics ; Stomach Neoplasms/*metabolism/pathology ; Support, Non-U.S. Gov't ; Urinary Plasminogen Activator/*biosynthesis/genetics