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 KhasaEmail author
Biotechnologically relevant enzymes and proteins


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.


Streptokinase 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.

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)


  1. Abad S, Kitz K, Hormann A, Schreiner U, Hartner FS, Glieder A (2010) Real-time PCR-based determination of gene copy numbers in Pichia pastoris. Biotechnol J 5:413–420. CrossRefPubMedGoogle Scholar
  2. Adivitiya, Dagar VK, Devi N, Khasa YP (2016) High level production of active streptokinase in Pichia pastoris fed-batch culture. Int J Biol Macromol 83:50–60. CrossRefGoogle Scholar
  3. Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98:5301–5317. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Ayed A, Rabhi I, Dellagi K, Kallel H (2008) High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris. Enzym Microb Technol 42(2):173–180. CrossRefGoogle Scholar
  5. Azoun SB, Belhaj AE, Kallel H (2016) Rabies virus glycoprotein enhanced expression in Pichia pastoris using the constitutive GAP promoter. Biochem Eng J 113:77–85. CrossRefGoogle Scholar
  6. Balagurunathan B, Ramchandra NS, Jayaraman G (2008) Enhancement of stability of recombinant streptokinase by intracellular expression and single step purification by hydrophobic interaction chromatography. Biochem Eng J 39:84–90. CrossRefGoogle Scholar
  7. Bera S, Thillai K, Sriraman K, Jayaraman G (2015) Process strategies for enhancing recombinant streptokinase production in Lactococcus lactis cultures using P170 expression system. Biochem Eng J 93:94–101. CrossRefGoogle Scholar
  8. Charoenrat T, Khumruaengsri N, Promdonkoy P, Rattanaphan N, Eurwilaichitr L, Tanapongpipat S, Roongsawang N (2013) Improvement of recombinant endoglucanase produced in Pichia pastoris KM71 through the use of synthetic medium for inoculum and pH control of proteolysis. J Biosci Bioeng 116:193–198. CrossRefPubMedGoogle Scholar
  9. Chaudhary A, Vasudha S, Rajagopal K, Komath SS, Garg N, Yadav M, Mande SC, Sahni G (1999) Function of the central domain of streptokinase in substrate plasminogen docking and processing revealed by site-directed mutagenesis. Protein Sci 8:2791–2805. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dagar VK, Khasa YP (2018) Combined effect of gene dosage and process optimization strategies on high-level production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris fed-batch culture. Int J Biol Macromol 108:999–1009. CrossRefGoogle Scholar
  11. Dagar VK, Adivitiya, Devi N, Khasa YP (2016) Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris. J Ind Microbiol Biotechnol 43:1373–1386. CrossRefGoogle Scholar
  12. Damasceno LM, Huang CJ, Batt CA (2012) Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 93(1):31–39. CrossRefPubMedGoogle Scholar
  13. Estrada MP, Hernandez L, Perez A, Rodriguez P, Serrano R, Rubiera R, Pedraza A, Padron G, Antuch W, de la FJ, Herrera L (1992) High level expression of streptokinase in Escherichia coli. Biotechnology (N Y) 10:1138–1142. CrossRefGoogle Scholar
  14. Goyal D, Sahni G, Sahoo DK (2009) Enhanced production of recombinant streptokinase in Escherichia coli using fed-batch culture. Bioresour Technol 100:4468–4474. CrossRefPubMedGoogle Scholar
  15. Han M, Wang X, Ding H, Jin M, Yu L, Wang J, Yu X (2014) The role of N-glycosylation sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris. Enzym Microb Technol 54:32–37. CrossRefGoogle Scholar
  16. Idiris A, Tohda H, Kumagai H, Takegawa K (2010) Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 86:403–417. CrossRefPubMedGoogle Scholar
  17. Ito K, Ishimaru T, Kimura F, Matsudomi N (2007) Importance of N-glycosylation positioning for secretion and folding of ovalbumin. Biochem Biophys Res Commun 361(3):725–731. CrossRefPubMedGoogle Scholar
  18. Jackson KW, Malke H, Gerlach D, Ferretti JJ, Tang J (1986) Active streptokinase from the cloned gene in Streptococcus sanguis is without the carboxyl-terminal 32 residues. Biochemistry 25:108–114. CrossRefPubMedGoogle Scholar
  19. Jahic M, Wallberg F, Bollok M, Garcia P, Enfors SO (2003a) Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures. Microb Cell Factories 2:6. CrossRefGoogle Scholar
  20. Jahic M, Gustavsson M, Jansen AK, Martinelle M, Enfors SO (2003b) Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. J Biotechnol 102(1):45–53. CrossRefPubMedGoogle Scholar
  21. Jia H, Guo Y, Song X, Shao C, Wu J, Ma J, Shi M, Miao Y, Li R, Wang D, Tian Z, Xiao W (2016) Elimination of N-glycosylation by site mutation further prolongs the half-life of IFN-alpha/Fc fusion proteins expressed in Pichia pastoris. Microb Cell Factories 15(209).
  22. Kobayashi K, Kuwae S, Ohya T, Ohda T, Ohyama M, Ohi H, Tomomitsu K, Ohmura T (2000) High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation. J Biosci Bioeng 89:55–61. CrossRefPubMedGoogle Scholar
  23. Kumar R, Singh J (2004) Expression and secretion of a prokaryotic protein streptokinase without glycosylation and degradation in Schizosaccharomyces pombe. Yeast 21:1343–1358. CrossRefPubMedGoogle Scholar
  24. Kunadian V, Gibson CM (2012) Thrombolytics and myocardial infarction. Cardiovasc Ther 30:e81–e88. CrossRefPubMedGoogle Scholar
  25. Kunamneni A, Abdelghani TT, Ellaiah P (2007) Streptokinase—the drug of choice for thrombolytic therapy. J Thromb Thrombolysis 23:9–23. CrossRefPubMedGoogle Scholar
  26. Laplace F, Muller J, Gumpert J, Malke H (1989) Novel shuttle vectors for improved streptokinase expression in streptococci and bacterial L-forms. FEMS Microbiol Lett 53:89–94. CrossRefPubMedGoogle Scholar
  27. Lee SJ, Evers S, Roeder D, Parlow AF, Risteli J, Risteli L, Lee YC, Feizi T, Langen H, Nussenzweig MC (2002) Mannose receptor-mediated regulation of serum glycoprotein homeostasis. Science 295:1898–1901. CrossRefPubMedGoogle Scholar
  28. Lee C, Lee S, Shin SG, Hwang S (2008) Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli. Appl Microbiol Biotechnol 78(2):371–376. CrossRefPubMedGoogle Scholar
  29. Lin LF, Oeun S, Houng A, Reed GL (1996) Mutation of lysines in a plasminogen binding region of streptokinase identifies residues important for generating a functional activator complex. Biochemistry 35:16879–16885. CrossRefPubMedGoogle Scholar
  30. Liu B, Shi D, Chang S, Gong X, Yu Y, Sun Z, Wu J (2015) Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast. Sci Rep 5(7678).
  31. Malke H, Gerlach D, Kohler W, Ferretti JJ (1984) Expression of a streptokinase gene from Streptococcus equisimilis in Streptococcus sanguis. Mol Gen Genet 196:360–363.
  32. Nakajima M, Koga T, Sakai H, Yamanaka H, Fujiwara R, Yokoi T (2010) N-glycosylation plays a role in protein folding of human UGT1A9. Biochem Pharmacol 79(8):1165–1172. CrossRefPubMedGoogle Scholar
  33. Pimienta E, Ayala JC, Rodriguez C, Ramos A, Van Mellaert L, Vallin C, Anne J (2007) Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans. Microb Cell Factories 6:20. CrossRefGoogle Scholar
  34. Pratap J, Rajamohan G, Dikshit KL (2000) Characteristics of glycosylated streptokinase secreted from Pichia pastoris: enhanced resistance of SK to proteolysis by glycosylation. Appl Microbiol Biotechnol 53(4):469–475. CrossRefPubMedGoogle Scholar
  35. Shi GY, Chang BI, Chen SM, Wu DH, Wu HL (1994) Function of streptokinase fragments in plasminogen activation. Biochem J 304(1):235–241. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Shi GY, Chang BI, Su SW, Young KC, Wu DH, Chang LC, Tsai YS, Wu HL (1998) Preparation of a novel streptokinase mutant with improved stability. Thromb Haemost 79(05):992–997. CrossRefPubMedGoogle Scholar
  37. Singh A, Upadhyay V, Panda AK (2015a) Solubilization and refolding of inclusion body proteins. Methods Mol Biol 1258: 283–291. Scholar
  38. Singh K, Shandilya M, Kundu S, Kayastha AM (2015b) Heat, acid and chemically induced unfolding pathways, conformational stability and structure-function relationship in wheat alpha-amylase. PLoS One 10:e0129203. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Sinha J, Plantz BA, Inan M, Meagher MM (2005) Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-τ. Biotechnol Bioeng 89:102–112. CrossRefPubMedGoogle Scholar
  40. Tsujikawa M, Okabayashi K, Morita M, Tanabe T (1996) Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast, Pichia pastoris and characterization of the secreted product. Yeast 12(6):541–553.<541::AID-YEA935>3.0.CO;2-A CrossRefPubMedGoogle Scholar
  41. Wang Z, Wang Y, Zhang D, Li J, Hua Z, Du G, Chen J (2010) Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation. Bioresour Technol 101:1318–1323. CrossRefPubMedGoogle Scholar
  42. Welfle H, Misselwitz R, Fabian H, Damerau W, Hoelzer W, Gerlach D, Kalnin NN, Venyaminov SY (1992) Conformational properties of streptokinase—secondary structure and localization of aromatic amino acids. Int J Biol Macromol 14:9–18. CrossRefPubMedGoogle Scholar
  43. Wong SL, Ye R, Nathoo S (1994) Engineering and production of streptokinase in a Bacillus subtilis expression-secretion system. Appl Environ Microbiol 60:517–523PubMedPubMedCentralGoogle Scholar
  44. Wu XC, Ye R, Duan Y, Wong SL (1998) Engineering of plasmin-resistant forms of streptokinase and their production in Bacillus subtilis: streptokinase with longer functional half-life. Appl Environ Microbiol 64:824–829PubMedPubMedCentralGoogle Scholar
  45. Wu DH, Shi GY, Chuang WJ, Hsu JM, Young KC, Chang CW, Wu HL (2001) Coiled coil region of streptokinase gamma-domain is essential for plasminogen activation. J Biol Chem 276:15025–15033. CrossRefPubMedGoogle Scholar
  46. Wu M, Shen Q, Yang Y, Zhang S, Qu W, Chen J, Sun H, Chen S (2013) Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115. J Ind Microbiol Biotechnol 40:589–599. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Xu C, Ng DTW (2015) Glycosylation-directed quality control of protein folding. Nat Rev Mol Cell Biol 16:742–752. CrossRefPubMedGoogle Scholar
  48. Yang M, Yu XW, Zheng H, Sha C, Zhao C, Qian M, Xu Y (2015) Role of N-linked glycosylation in the secretion and enzymatic properties of Rhizopus chinensis lipase expressed in Pichia pastoris. Microb Cell Factories 14(1):40. CrossRefGoogle Scholar
  49. Zhou X, Yu Y, Tao J, Yu L (2014) Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation. J Biosci Bioeng 118:420–425. CrossRefPubMedGoogle Scholar
  50. Zou S, Huang S, Kaleem I, Li C (2013) N-glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris. J Biotechnol 164:75–81. CrossRefPubMedGoogle Scholar

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
    Email author
  1. 1.Department of MicrobiologyUniversity of Delhi South CampusNew DelhiIndia

Personalised recommendations