title: A comparative study on the activity and antigenicity of truncated and full-length forms of streptokinase.
abstract: Application of streptokinase (SK) as a common and cost-effective thrombolytic drug is limited by its antigenicity and undesired hemorrhagic effects. Prior structural/functional and epitope-mapping studies on SK suggested that removal of 59 N-terminal residues led to its fibrin dependency and identified SK antigenic regions, respectively. Following in silico analyses two truncated SK proteins were designed and compared for their fibrin specificity and antigenicity with the full-length SK. Computer-based modeling was used to predict the effect of vector (pET41a)-born protein tags on the conformation of SK fragments. SK60-386, SK143-386 and full-length SK (1-414) were separately cloned, expressed in BL21 E. coli cells and confirmed by Western-blotting. Functional activity of the purified proteins was evaluated with chromogenic and clot lysis assays and their antigenicity was tested by ELISA assay using rabbit anti-streptokinase antibody. As expected, chromogenic bioassay showed a major activity decline for SK60-386 and SK143-386 (83 and 91 percent, respectively), compared to SK1-414. However, in clot lysis assay, which is a fibrin-dependent pharmacopoeia-approved test, SK60-386 and SK143-386 were respectively 35 and 31 percent more active though lysed the clots slower than full-length SK. Antigenic analysis also indicated significant decrease in ELISA signals obtained for truncated proteins compared to SK1-414 (45 and 28 percent less reactivity for SK143-386 and SK60-386, respectively, p < 0.0001). The results of this study for the first time pointed to SK143-386 and SK60-386, as improved SK derivatives with increased fibrin-selectivity and decreased antigenicity as well as acceptable bioactivity profiles in a pharmacopoeia-based analysis, which deserve more detailed pharmacological studies.
authors.affiliation: Hepatitis and AIDS Dept, Pasteur Institute of Iran, Tehran.
authors.forename: Reza
authors.initials: R
authors.lastname: Arabi
authors.forename: Farzin
authors.initials: F
authors.lastname: Roohvand
authors.forename: Daryoush
authors.initials: D
authors.lastname: Norouzian
authors.forename: Soroush
authors.initials: S
authors.lastname: Sardari
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Aghasadeghi
authors.forename: Hosein
authors.initials: H
authors.lastname: Khanahmad
authors.forename: Arash
authors.initials: A
authors.lastname: Memarnejadian
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Motevalli
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013300:Streptokinase / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D013300:Streptokinase