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Applied Microbiology and Biotechnology

, Volume 102, Issue 24, pp 10561–10577 | Cite as

Engineering of deglycosylated and plasmin resistant variants of recombinant streptokinase in Pichia pastoris

  • Adivitiya
  • Babbal
  • Shilpa Mohanty
  • Yogender Pal Khasa
Biotechnologically relevant enzymes and proteins
  • 145 Downloads

Abstract

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.

Keywords

Streptokinase Pichia pastoris qPCR N-glycosylation Proteolysis Plasmin resistance 

Notes

Acknowledgements

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.

Ethical statement

This work does not involve any human and animal studies.

Supplementary material

253_2018_9402_MOESM1_ESM.pdf (820 kb)
ESM 1 (PDF 820 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Adivitiya
    • 1
  • Babbal
    • 1
  • Shilpa Mohanty
    • 1
  • Yogender Pal Khasa
    • 1
  1. 1.Department of MicrobiologyUniversity of Delhi South CampusNew DelhiIndia

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