Abstract:

The cloning of Retep lase ( r-PA) gene of tissue-type plasminogen activator ( t-PA) mutant was first carried out, and the gene was extracellularly expressed in Pichia methanolica. Next, the gene-encoding Reteplase ( r-PA) was cloned to pMD18-T vector by PCR using recombinant strain Trichoderma reesei 306 chromosome DNA as the template. Then, a sequence determination was conducted, indicating that the cloned gene sequence is of a length of 1. 1 kb and an identity of 99. 91% or 100% respectively with the published Reteplase gene or amino acids. Moreover, the secreted expression plasmid pMETαA-r-PA of Pichia methanolica was not only constructed and digested with PacⅠ but also transformed into Pichia methanolica PMAD16 by means of electroporation. The positive transformants that integrate r-PA genes in their genomes were thus obtained, which was verified by PCR technique and Mut phenotype determination. After the shake-flask culture and the induced expression with methanol as the only carbon source, the extracellular Reteplase ( r-PA) activity was up to 27. 93mol/ ( s·L).

Key words: tissue-type plasminogen activator, Pichia methanolica, Retep lase ( r2PA), expression