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Iterative optimization of high-affinity proteases inhibitors using phage display. 1. Plasmin
1Protein Engineering Corporation, Cambridge, Massachusetts 02138, USA.
Biochemistry
|June 18, 1996
Summary
We engineered phage display libraries to create potent inhibitors of human plasmin (PLA). The best variant, EPI-P302, exhibits extremely high affinity and specificity, offering a stable, human-based therapeutic candidate.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Lipoprotein-associated coagulation inhibitor (LACI-D1) is a Kunitz domain protein.
- Phage display is a powerful technique for protein engineering and selection.
Purpose of the Study:
- To engineer high-affinity, specific inhibitors of human plasmin (PLA) using phage display.
- To develop a novel therapeutic candidate for conditions involving PLA activity.
Main Methods:
- Generation of iterative phage display libraries (Lib#1, Lib#2, Lib#3) with variants of LACI-D1.
- Screening libraries against immobilized human plasmin (PLA) to select for high-affinity binders.
- Characterization of selected protein variants for inhibitory activity, specificity, and stability.
Main Results:
- Engineered protein EPI-P211 inhibited PLA with a Ki of 2 nM, a 500-fold improvement over LACI-D1.
- Further optimization yielded EPI-P302 with a Ki of 87 pM, over 12,500-fold better than LACI-D1.
- EPI-P302 demonstrated high specificity for PLA and stability under various conditions.
Conclusions:
- Phage display is effective for generating highly potent and specific protein inhibitors.
- The engineered protein EPI-P302 represents a promising candidate for therapeutic applications targeting PLA.
- This approach allows for the creation of stable, human-based binding molecules with tailored properties.