Iranian Biomedical Journal
مجله بیومدیکال ایران
IBJ
Basic Sciences
http://ibj.pasteur.ac.ir
1
admin
1028-852X
2008-823X
-
10.61882/ibj
-
8888
-
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
Related Fields
Related Fields
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