Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris
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Streptokinase, a therapeutically important thrombolytic agent, is prone to C-terminal degradation and plasmin-mediated proteolytic processing. Since the protein was glycosylated during secretion from Pichia pastoris, therefore, the role of carbohydrate moieties on its stability was analyzed via in vivo blocking of N-glycosylation using tunicamycin where an increased degradation of streptokinase was observed. Further, the in vitro site-directed mutagenesis of the three putative N-glycosylation sites at asparagine residues 14, 265, and 377 to alanine revealed the essentiality of glycosylation of the 14th amino acid residue in its post-translational proteolytic stability without significantly affecting its biological activity. However, the mutation of both Asn265 and Asn377 did not seem to contribute toward its glycosylation but resulted in a 39% lower specific activity in case of the rSK-N265,377A. Moreover, the mutation of all three glycosylation positions drastically reduced the secretory expression of native streptokinase from 347 to 186.6 mg/L for the triple mutant with a 14% lower specific activity of 56,738 IU/mg from 65,808 IU/mg. The secondary structure, tertiary structure, and thermal transition point (45–55 °C) of all the deglycosylated variants did not show any significant differences when compared with fully glycosylated native streptokinase using CD and fluorescence spectroscopy. Furthermore, the longer acting plasmin-resistant variants were also developed via the mutation of lysine residues 59 and 386 to glutamine which enhanced its biological stability as a ~ 1.5-fold increase in the caseinolytic zone size was observed in case of rSK-K59Q and also in rSK-K59,386Q mutant without affecting the structural properties.
KeywordsStreptokinase Pichia pastoris qPCR N-glycosylation Proteolysis Plasmin resistance
The financial support from University of Delhi through R&D grant to Dr. Y. P. Khasa is sincerely acknowledged. Adivitiya and Babbal are the recipients of research fellowship from the Council of Scientific and Industrial Research (CSIR), Govt. of India, New Delhi. The authors would also like to acknowledge the generous technical help of Dr. Sanjay Kumar Dey and Prof. Suman Kundu (Department of Biochemistry, UDSC, New Delhi) during thermal fluorescence analysis.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interest.
This work does not involve any human and animal studies.
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