title: High level production of active streptokinase in Pichia pastoris fed-batch culture.
abstract: Streptokinase is a biological macromolecule involved in dissolution of fibrin blood clot and favourably used in various clinical applications. This protein is poorly expressed in soluble form due to its toxic effects on host physiology. The extracellular expression of recombinant streptokinase (SK) with and without 6xHis tag was obtained by cloning its gene under the α-mating factor signal sequence and alcohol inducible AOX1 promoter. Host-vector combinations were optimized to select a hyper producer. From shake flask optimization studies, a maximum expression of 582 mg/L of rSK (non-tagged) and 538 mg/L of rSK-His (His-tagged) protein was obtained when cells were induced at OD600 of 20. The high cell density fermentation increased the volumetric product concentration of rSK-His to a level of 4.25 g/L with a 7.9 folds increase from shake flask results. The specific product yield (YP/X) was 49.75 mg/g DCW along with a high volumetric productivity of 57.43 mg/L/h. The protein was predicted to have 15.43% α-helix and 26.43% β-sheet with tryptophan emission maxima of around 347 nm. The highest specific activity of rSK-His was 64,903 IU/mg with 1.48 folds purification whereas specific activity of rSK was 55,240 IU/mg with 1.22 folds purification.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110 021, India.
authors.lastname: Adivitiya
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110 021, India.
authors.forename: Vikas Kumar
authors.initials: VK
authors.lastname: Dagar
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110 021, India.
authors.forename: Nirmala
authors.initials: N
authors.lastname: Devi
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110 021, India. Electronic address: yogi110@gmail.com.
authors.forename: Yogender Pal
authors.initials: YP
authors.lastname: Khasa
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D005285:Fermentation / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D000072235:Mating Factor; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013300:Streptokinase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D010455:Peptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000072235:Mating Factor; D013300:Streptokinase
keywords: AOX1 promoter; Fed-batch fermentation; Glycosylation; Pichia pastoris; Secretory expression; Streptokinase