Purification and characterization of recombinant Streptokinase expressed in E.coli from Streptococcus equisimilis with N-terminal methionine

Abstract

Streptokinase is a extracellular enzyme which is extracted from strains of beta Hemolytic streptococcus. The enzyme is a non-protease plasminogen activator that activates plasminogen to plasmin and degrades fibrin clot through its specific lysine binding site which is used in thrombolytic therapy. Purification of streptokinase produced from S.equisimilis in E.coli with N-terminal methionine was carried out in 3 Chromatography purification steps, 1) CM-Sepharose-FF at pH 4.2 followed by concentration and dialysis over night with Tris-HCl pH 8.0. Partially purified dialyzed enzyme sample was loaded on to 2) DEAE-Sepharose-FF column. The Purified fractions of DEAE column were pooled and applied on to Sephadex G-100 column. Enzyme purity was confirmed by SDS-PAGE and RP-HPLC.Its biological activity is determined by specific streptokinase assay and characterised the enzyme by Peptide mapping, MALDI-TOF, Isoelectric-focusing and RP-HPLC. The isoelectric point (pI) of streptokinase is around 4.98.The results of characterization shows that it contains two forms (Isomers) of streptokinase expressed in E. coli which was analyzed by RP-HPLC and chromogenic assay. The variation is formed by isomer-1 in which 85% of Streptokinase expressed without methionine (85000IU/mg) and Isomer-2 in which 15% of streptokinase expressed with methionine (nil activity) in E. coli. This phenomenon shows that the presence and absence of methionine in isomers of streptokinase varying the catalytic activity of the enzyme.

Role of Fibrinolytic Enzymes in Anti-Thrombosis Therapy

“…Fermented soybeans have been consumed as traditional foods in many Asian countries, and indeed, they are sources of nattokinase and other fibrinolytic enzymes. These includes natto, a Japanese traditional fermented soybean obtained from fermentation with Bacillus subtilis G8 and Bacillus subtilis (Chang et al, 2012;Lucy et al, 2019), moromi, oncom and gambus (Indonesian traditional fermented soybean obtained from fermentation with Bacillus cereus, Bacillus subtilus, Bacillus pumilus, and Stenotrophomonas sp, respectively) (Afifah et al, 2014;Nailufar et al, 2016;Stephani et al, 2017;Syahbanu et al, 2020) as well as douchi and doufuru (Chinese traditional fermented soybean obtained from fermentation with Bacillus (Ok and Choi, 2005;Wang et al, 2006;Jo et al, 2011;Wei et al, 2011;Yuan et al, 2012;Chen et al, 2013;Heo et al, 2013;Park et al, 2013;Gad et al, 2014;Thokchom and Joshi, 2014;Bi et al, 2015;Huy et al, 2016;Liu et al, 2016;Nailufar et al, 2016;Hu et al, 2019;Ito, 2020;Syahbanu et al, 2020 (Shirasaka et al, 2012;Biji et al, 2016;Vijayaraghavan et al, 2016a;Vijayaraghavan et al, 2016b;Vijayaraghavan et al, 2019), seafood fermented with Bacillus sp, Bacillus. velezensis BS2, Bacillus pumilus BS15 and Bacillus coagulans (Anh et al, 2015;Prihanto and Firdaus, 2019;Yao et al, 2019;Kim et al, 2020) and liquors obtained from fermentation with Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus-Genus (Lapsongphon et al, 2013;Johnson e...…”

Thrombolytic Enzymes of Microbial Origin: A Review

“…SK is isolated and derived from several strains of β-hemolytic Streptococci of Lancefield group A, C, and G [ 126 , 127 ]. The Streptococcus equisimilis GCS strain H46A and group C have been chiefly used for SK production [ 128 ]. SK comprises three structural domains: α, β, and γ at the 1–146, 147–290, and 291–414 AA positions catenated by two flexible coil regions.…”