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Updated: Jun 28, 2026
Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
Published on: January 22, 2014
High yielding recombinant Staphylokinase in bacterial expression system--cloning, expression, purification and
Nagnath Mandi1, Sudheerbabu Soorapaneni, Sachin Rewanwar
1Biotechnology R & D, Lupin Limited, 46A/47A, Nande Village, Mulshi Taluka, Lupin Research Park, Pune-411042, India. nagnathmandi@lupinpharma.com
Abstract:
Staphylokinase (SAK) is emerging as an important thrombolytic agent. In this report, we describe the cloning, expression, purification and activity studies of the SAK gene of Staphylococcus aureus from a custom synthesised SAK gene. The SAK gene of 411 bp yielded a protein of approximately 15 kDa when expressed under pET21a vector using IPTG as an inducer in BL21 (DE3) pLysE codon Plus cells. The recombinant SAK (rSAK) was soluble in nature and constituted nearly 35% of the total cellular protein as estimated by densitometry scanning. Fermentation studies were carried out to optimize various parameters for maximizing the yield of rSAK and with the optimized medium, the yield of rSAK was nearly 2.8 g/L of fermentation broth, which is highest yield of rSAK expressed in any bacterial system till date. Two simple purification steps of ion-exchange chromatography yielded homogenous rSAK with almost 36% recovery. The purified SAK protein was characterized by MALDI-TOF and by plasminogen activation studies. The rSAK was found to be active by the chromogenic substrate assay method.
