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Published on: July 29, 2007
Purification and characterization of mutant miniPlasmin for thrombolytic therapy
Xiaotao Lin1, Yan Wang, Yanwen Zhang
1Genecopoeia Inc, 9620 Medical Center Drive #101, 20850, Rockville, MD, USA. shinlilin@yahoo.com.
Background:
Previous animal studies by us and others have indicated that catheter-administered plasmin or its des-kringle derivatives may be more appropriate alternatives to plasminogen activators for treating thrombolytic diseases, since it has a very short serum half-life and therefore does not result in hemorrhaging. We have previously produced recombinant miniPlasmin (mPlasmin) that was proven suitable for treating peripheral arterial occlusion in animal models. However, our previous results showed that non-specific cleavage at position K698 of mPlasmin during activation hindered the further development of this promising therapeutic candidate. In order to minimize or eliminate the non-specific cleavage problem, we performed saturation mutagenesis at the K698 position to develop a mutant form of mPlasmin for thrombolytic therapy.
Methods:
We changed K698 to 16 other amino acids, with preferred E. coli codons. Each of these mutants were expressed in E. coli as inclusion bodies and then refolded, purified, and subsequently characterized by detailed kinetic assays/experiments/studies which identified highly active mutants devoid of non-specific cleavage.
Results:
Activation studies indicated that at those conditions in which the wild type enzyme is cut at the non-specific position K698, the active mutants can be activated without being cleaved at this position.
Conclusions:
From the above results, we selected two mutants, K698Q and K698N, as our lead candidates for further thrombolytic drug developments. The selected mutants are potentially better therapeutic candidates for thrombolytic therapy.
Insights
Researchers developed a modified miniPlasmin (mPlasmin) to improve thrombolytic therapy. Mutating a specific site prevented unwanted cleavage, creating promising drug candidates for treating blood clots.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Catheter-administered plasmin and derivatives show promise for thrombolytic diseases due to short half-lives and reduced hemorrhage risk.
- Recombinant miniPlasmin (mPlasmin) has demonstrated efficacy in animal models for peripheral arterial occlusion.
- Non-specific cleavage at the K698 position of mPlasmin has previously hindered its therapeutic development.
Purpose of the Study:
- To engineer a mutant form of mPlasmin by addressing the non-specific K698 cleavage site.
- To develop a more effective and safer therapeutic agent for thrombolytic therapy.
Main Methods:
- Saturation mutagenesis was performed at the K698 position of mPlasmin, substituting it with 16 different amino acids.
- Mutants were expressed in E. coli, refolded, purified, and characterized using kinetic assays.
- Activation studies were conducted to evaluate cleavage patterns compared to wild-type mPlasmin.
Main Results:
- Several highly active mPlasmin mutants were identified that were devoid of non-specific cleavage at K698.
- The active mutants could be activated without cleavage at the K698 position, unlike the wild-type enzyme.
- Mutants K698Q and K698N demonstrated robust activity and specificity.
Conclusions:
- The K698Q and K698N mPlasmin mutants are identified as lead candidates for further thrombolytic drug development.
- These modified mPlasmin variants represent potentially improved therapeutic options for thrombolytic therapy.
- Minimizing non-specific cleavage enhances the suitability of mPlasmin for clinical applications.
