A fast-acting, modular-structured staphylokinase fusion with Kringle-1 from human plasminogen as the fibrin-targeting domain offers improved clot lysis efficacy.
To develop a fast-acting clot dissolving agent, a clot-targeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein in Bacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced from B. subtilis was improperly folded because of its complicated disulfide-bond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flow-cell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues.
Wu
Sau-Ching
SC
Division of Molecular, Cellular, and Microbial Biology, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
Castellino
Francis J
FJ
Wong
Sui-Lam
SL
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005337:Fibrin / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010477:Perfusion; D010843:Pichia / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D008024:Ligands; D011993:Recombinant Fusion Proteins; D013912:Threonine; D005337:Fibrin; D010958:Plasminogen; D004259:DNA-Directed DNA Polymerase; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus