Production of Stable Serum Albumin Fused Streptokinase in Pichia pastoris
Production of Stable Serum Albumin Fused Streptokinase in Pichia pastoris
Saad Tahir, Nadeem Ahmed*, Mohsin Ahmad Khan, Muhammad Akram, Rabia Abbas and Kausar Malik
ABSTRACT
Streptokinase (SK) is a thrombolytic and fibrinolytic protein (47kDa), naturally produced by beta-hemolytic streptococci. Purified SK is used for many blood circulatory complications, i.e., myocardial infarction, ischemic stroke and pulmonary embolism. In this study, human albumin fusion technology has been developed to increase the half-life in-vivo and also invoke less immune response. We designed codon-optimized HSA-SK fusion gene, integrated into -pPICZaB vector, cloned and transferred into Pichia pastoris strain GS115. The transformants showing highest resistance for Zeocin™ was selected for protein expression which produced to >350mg/L HSA-SK fusion protein. Further, affinity chromatography was carried out for the purification of the HSA-SK fusion protein. SDS-PAGE, western blot, and RP-HPLC analysis were also conducted, which confirmed the 98% purity of the product with 40% yeild having a molecular weight of almost 111kDa. The purified fusion protein (HSA-SK) was also verified via western blotting by using anti-streptokinase antibodies. Clot lysis assay was used to estimate the biological activity of HSA-SK. For half-life estimation, proteolytic and thermal stability of HSA-SK was also checked.
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