Purification and characterization of mutant miniPlasmin for thrombolytic therapy
| Autor: | Lin Xiaotao, Wang Yan, Zhang Yanwen, Huang Bing, Lin James J, Hallock Scott J, Yu Hong, Shao Hongwei, Yan Jing, Huang Bo, Zhang Xuejun C, Cao Wei, Xu Xueming, Lin Xinli |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: | |
| Zdroj: | Thrombosis Journal, Vol 11, Iss 1, p 2 (2013) |
| Druh dokumentu: | article |
| ISSN: | 1477-9560 47578025 |
| DOI: | 10.1186/1477-9560-11-2 |
| Popis: | Abstract 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. |
| Databáze: | Directory of Open Access Journals |
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