Pilot-scale production and purification of a staphylokinase-based fusion protein over-expressed in Escherichia coli

SFH, a recombinant staphylokinase-based fusion protein linked by the factor Xa recognition peptide at the N-terminus of hirudin, is a promising therapeutic candidate for thromboembolic diseases. To develop SFH into a new thrombolytic agent, scaled-up production was carried out to provide sufficient preparation for animal safety and clinical studies. Here, we describe a pilot-scale cultivation and purification process for the production of SFH. A high-cell-density fed-batch cultivation for the production of SFH in E. coli was developed in a 40-L bioreactor, which produced about 1.1 g/L of recombinant protein. SFH was purified to homogeneity from the E. coli lysate by expanded bed adsorption chromatography and anion-exchange chromatography, with over 99% purity and 54% recovery. Moreover, the residual endotoxin content was less than 0.5 EU/mL. The molecular weight and in vitro bioactivity of SFH were also determined by electrospray ionization-mass spectrometry (ESI-MS) and fibrinolytic activity assay, respectively.