OVERPRODUKSI DAN PURIFIKASI STREPTOKINASE K59Q-K386Q, SERTA UJI KESTABILAN TERHADAP PEMOTONGAN OLEH PLASMIN MENGGUNAKAN ANALISIS WESTERN BLOT

• Main Author: Widayanti, Aniek
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/72699
• Institution: Institut Teknologi Bandung

Streptokinase remains as a drug of choice for stroke and acute myocardial infarction therapy because of low cost compared to other fibrinolytic agents. The short half-life of streptokinase dues to cleavage by plasmin that cleaves peptide bond after lysine residu. Mutations in the open reading frame of streptokinase on codon encoding lysine at residues 59 and 386 were done (ska K59Q-K386Q), and were cloned to an expression vector pET- 32b. This research was aimed to produce streptokinase with improved stability to plasmin cleavage and to develop polyclonal antibody anti-streptokinase for analysis of streptokinase using Western blot. Vector pET-32b ska K59Q-K386Q was transformed into E. coli BL21 and was characterized. Overproduction was done using IPTG 0.5 mM as inducer at three hours incubation. Ska mutein 2 was successfully overproduced, confirmed by the presence of a thick band at 64.42 kDa in polyacrylamide gel. Purification of Ska mutein 2 was optimized with various concentrations of imidazole using nickel affinity chromatography and its concentration was determined using Bradford assay. Polyclonal antibody anti-streptokinase was successfully developed in rabbit strain japanese white for 30 days and was analyzed using dot blot and Western blot. Stability assay of Ska 2 mutein was done by incubated Ska 2 mutein with human plasma and was analyzed using Western blot. Stability assay of Ska 2 mutein against plasmin cleavage showed that Ska mutein 2 was recognized by plasmin.