Expression and intein-mediated purification of novel staphylokinase SakSTAR with reduced immunogenicity and antiplatelet and antithrombin activation.
In an effort to combine the benefits of fibrinolytics, such as staphylokinase (Sak), with those of thrombin inhibitors for the prevention of vessel reocclusion after vascular injury, we produced chimeric protein with plasminogen activator and thrombin-inhibiting properties. This fusion protein was a construct consisting of Sak (SakSTAR) lengthened about 36 amino acids from the C-terminus end of hirudin. We inserted 16 point mutations into the sequence of the gene encoding SakSTAR for reduced antibody binding from 50% to about 17% and inserted two RGD sequences for antiplatelet activity. The inhibition rate of platelet aggregation was 27%. Moreover, we proposed an efficient method of expression and purification in which we used 16 mg/L of anEscherichia coli strain of this novel fusion protein and retained full biologic activities toward plasminogen and thrombin.
Kochanowski
Rafal
R
Department of Food Chemistry, Technology and Biotechnology, Chemical Faculty, Gdansk University of Technology, 11/12 Narutowicza St., Gdansk, 80-952, Poland. rkoch@chem.pg.gda.pl
Kotlowski
Roman
R
Szweda
Piotr
P
D000595:Amino Acid Sequence; D000925:Anticoagulants / Q000737:chemistry; D000991:Antithrombins / Q000737:chemistry*; D001792:Blood Platelets / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005337:Fibrin / Q000737:chemistry; D005342:Fibrinolysis; D006629:Hirudins / Q000737:chemistry; D006801:Humans; D047668:Inteins; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D010960:Plasminogen Activators / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry*; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors
D000925:Anticoagulants; D000991:Antithrombins; D006629:Hirudins; D010975:Platelet Aggregation Inhibitors; D011993:Recombinant Fusion Proteins; D005337:Fibrin; D010960:Plasminogen Activators; D013917:Thrombin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus