title: High-level expression of non-glycosylated and active staphylokinase from Pichia pastoris.
abstract: Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (approximately 1 g/l) of extracellular glycosylated rSAK (approximately 18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (approximately 15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.
authors.affiliation: Biotechnology R & D, Lupin Limited, 46A/47A, Nande Village, Mulshi Taluka, Pune 411042, India.
authors.forename: Anjali
authors.initials: A
authors.lastname: Apte-Deshpnade
authors.forename: Goutam
authors.initials: G
authors.lastname: Mandal
authors.forename: Sudheerbabu
authors.initials: S
authors.lastname: Soorapaneni
authors.forename: Bhaskarjyoti
authors.initials: B
authors.lastname: Prasad
authors.forename: Jitendra
authors.initials: J
authors.lastname: Kumar
authors.forename: Sriram
authors.initials: S
authors.lastname: Padmanabhan
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange / Q000379:methods; D006031:Glycosylation; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D010958:Plasminogen / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010958:Plasminogen; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus