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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.

journal_name

Appl Microbiol Biotechnol

journal_title

Applied microbiology and biotechnology

authors

Adivitiya,Babbal,Mohanty S,Khasa YP

doi

10.1007/s00253-018-9402-x

subject

Has Abstract

pub_date

2018-12-01 00:00:00

pages

10561-10577

issue

eissn

0175-7598

issn

1432-0614

pii

10.1007/s00253-018-9402-x

journal_volume

102

pub_type

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