Iranian Biomedical Journal
مجله بیومدیکال ایران
IBJ
Basic Sciences
http://ibj.pasteur.ac.ir
1
admin
1028-852X
2008-823X
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10.61186/ibj
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8888
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en
jalali
1398
10
1
gregorian
2020
1
1
24
1
online
1
fulltext
en
Contribution of Streptokinase-Domains from Groups G and A (SK2a) Streptococci in Amidolytic/Proteolytic Activities and Fibrin-Dependent Plasminogen Activation: A Domain-Exchange Study
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مقاله کامل
Full Length/Original Article
<strong>Background:</strong> Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SKg). Based on the variable region of SKβ, GAS-SKs (<em>ska</em>) are clustered as SK1 and SK2 (including cluster2-streptokinase (SK2a)/SK2b), which show low and high fibrinogen (FG)-dependent plasminogen (Plg) activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SK (<em>skcg</em>) variants display properties similar to SK1. Herein, by SKβ exchange between GGS (G88) and GAS-SK2a (STAB902) variants, the potential roles of SK domains in amidolytic/proteolytic activity and FG-bound-Plg activation are represented. <strong>Methods:</strong> Two parental SK<sub>G88</sub> and SK<sub>STAB902 </sub>genes were cloned into the <em>Nde</em>I/<em>Xho</em>I site of pET26b expression vector. The two chimeric SKβ-exchanged constructs (SK<sub>C1</sub><span dir="RTL">:</span> α<sub>G88</sub>-β<sub>STAB</sub>-γ<sub>G88</sub> and SK<sub>C2</sub>; α<sub>STAB</sub>-β<sub>G88</sub>-γ<sub>STAB</sub>) were constructed by <em>BstE</em>II/<em>BsiW</em>I digestion/cross-ligation in parental plasmids. SK were expressed in <em>E. coli</em> and purified by nickel-nitriloacetic acid chromatography. PA potencies of SKs were measured by colorimetric assay. <strong>Results:</strong> SDS-PAGE and Western-blot analyses confirmed the proper expression of 47-kDa SKs. Analyses indicated that the catalytic efficiency (K<sub>cat</sub>/K<sub>m</sub>) for amidolytic and proteolytic activity were less and moderately dependent on SKβ, respectively. The increase of FG-bound-Plg activation for SK<sub>STAB902</sub>/SK<sub>C1</sub> containing SK2aβ was around six times, whereas for SK<sub>G88</sub>/SK<sub>C2</sub> containing skcgβ, it was four times. <strong>Conclusion:</strong> Although SKβ has noticeable contribution in FG-bound-Plg activation activity, it had minor contribution in fibrin-independent, amidolytic activity. These data might be of interest for engineering fibrin-specific versions of SK.
Plasminogen, Streptokinase, Thrombolytic therapy
15
23
http://ibj.pasteur.ac.ir/browse.php?a_code=A-10-1-792&slc_lang=en&sid=1
Maryam
Rafipour
100319475328460054597
100319475328460054597
No
Department of Virology, Pasteur Institute of Iran, Tehran, Iran
Malihe
Keramati
100319475328460054598
100319475328460054598
No
Department of Nanobiotechnology, Pasteur Institute of Iran, Tehran, Iran
Mohammad Mehdi
Aslani
100319475328460054599
100319475328460054599
No
Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran
Arash
Arashkia
100319475328460054600
100319475328460054600
No
Department of Virology, Pasteur Institute of Iran, Tehran, Iran
Farzin
Roohvand
100319475328460054601
100319475328460054601
Yes
Department of Virology, Pasteur Institute of Iran, Tehran, Iran