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A fast-acting, modular-structured staphylokinase fusion with Kringle-1 from human plasminogen as the fibrin-targeting domain offers improved clot lysis efficacy.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2003 May 16; Vol. 278 (20), pp. 18199-206. Date of Electronic Publication: 2003 Mar 19. - Publication Year :
- 2003
-
Abstract
- 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.
- Subjects :
- Amino Acid Sequence
Bacillus subtilis metabolism
Binding Sites
Calorimetry
Cloning, Molecular
DNA-Directed DNA Polymerase metabolism
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
Fibrin chemistry
Fibrin metabolism
Glycosylation
Humans
Ligands
Metalloendopeptidases metabolism
Models, Molecular
Molecular Sequence Data
Perfusion
Pichia metabolism
Plasmids metabolism
Plasminogen metabolism
Protein Binding
Protein Structure, Tertiary
Recombinant Fusion Proteins chemistry
Threonine chemistry
Time Factors
Metalloendopeptidases chemistry
Plasminogen chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 278
- Issue :
- 20
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 12646571
- Full Text :
- https://doi.org/10.1074/jbc.M210919200