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Rivista di Biologia Molecolare e Biotecnologie
Indexed/Abstracted in: EMBASE, Science Citation Index Expanded (SciSearch), Scopus
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Minerva Biotecnologica 1998 Dicembre;10(4):174-77
Hydrophobic interaction chromatography applied to purification of recombinant streptokinase
Pérez N. 1, Urrutia E. 1, Camino J. 1, Orta D. R. 1, Torres Y. 1, Martinez Y. 2, Cruz M. 2, Alburquerque S. 1, Gil M. R. 1, Hernandez I. 1
1 Streptokinase Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba;
2 Quality Control Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba
Background. Recombinant streptokinase (rSk) is a streptococcal protein cloned in E. coli.11 Several methods have been described for streptokinase purification: ion exchange chromatography,12 affinity chromatography with canine plasmin13 and chromatography on immobilized acylated human plasminogen.14 Monoclonal antibodies anti-rSk immobilized to Sepharose15 have been used too. Recently this protein was purified using HIC.
Methods. rsk (CIGB, Cuba) was produced by fermentation strain K12 of E. coli,11 the protein was recovered after washed pellet, cellular disruption and solubilization. Several purification assays were done using TSK-Butyl (Tosohaas, Japan) as a support for hydrophobic interaction chromatography (HIC). The protein was loaded to the column with 1 M of ammonium sulfate before being washed using an elution gradient from 0.5 to 0 M of ammonium sulfate, in order to determine the elution point of the rSk.
Results. We could determine that this protein is partly hydrophobic, this determination was shown by analysis of its aminoacidic sequence. This protein has 415 aminoacids of which 36% are non polar. The absorption capacity for TSK Butyl 650 S varies from 15 to 20 mg/mL. The optimun elution point was obtained using 0.25 M of ammonium sultate, the eluted material was obtained with a high level of purity (<1% of contaminants). The recovery of rSk was about 49% using the mean of five assays.
Conclusions. The experimental process evaluated could be efficiently inserted in a downstream process to obtain recombinant streptokinase highly purified as final preparation and good recovery.