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

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..

Medienart:

E-Artikel

Erscheinungsjahr:

2018

Erschienen:

2018

Enthalten in:

Zur Gesamtaufnahme - volume:102

Enthalten in:

Applied microbiology and biotechnology - 102(2018), 24 vom: 08. Okt., Seite 10561-10577

Sprache:

Englisch

Beteiligte Personen:

Mohanty, Shilpa [VerfasserIn]
Khasa, Yogender Pal [VerfasserIn]

Links:

Volltext [lizenzpflichtig]

BKL:

58.30$jBiotechnologie

42.30$jMikrobiologie

Themen:

N-glycosylation
Plasmin resistance
Proteolysis
QPCR
Streptokinase

RVK:

RVK Klassifikation

Anmerkungen:

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

doi:

10.1007/s00253-018-9402-x

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

OLC2091137170

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