Construction and characterization of novel staphylokinase variants with antiplatelet aggregation activity and reduced immunogenecity.
To develop target thrombolytic agents with fibrinolytic activity, antiplatelet aggregation activity and reduced immunogenicity, two staphylokinase variants containing Arg-Gly-Asp (RGD) motif were constructed. Gene expression was induced in E. coli JF1125 and the variants, designated DGR and RL1, were purified with gel filtration and ion-exchange chromatography and the purity was over 95%. The fibrinolytic activity and kinetic constants of the two variants were comparable to those of recombinant wild-type staphylokinase. Both the variants can inhibit the platelet aggregation at a final concentration of 2 microM. The titers of antibodies against variants were much lower than those against recombinant staphylokinase in guinea pigs, which indicated that the immunogenicity of the variants was greatly reduced. These results confirm that it is possible to design and produce a bifunctional protein that possesses fibrinolytic and antiplatelet aggregation activities.
Su
Hua-Bo
HB
Department of Molecular Genetics, Shanghai Medical School, Fudan University, Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai 200032, China.
Zhang
Yu-Gao
YG
He
Jin-Tian
JT
Mo
Wei
W
Zhang
Yan-Ling
YL
Tao
Xian-Mei
XM
Song
Hou-Yan
HY
D000818:Animals; D000906:Antibodies / Q000097:blood / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods; D005343:Fibrinolytic Agents / Q000097:blood / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014644:Genetic Variation; D006168:Guinea Pigs; D007125:Immunogenetics / Q000379:methods; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010975:Platelet Aggregation Inhibitors / Q000097:blood / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship
D000906:Antibodies; D005343:Fibrinolytic Agents; D010975:Platelet Aggregation Inhibitors; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus