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Purification and characterization of mutant miniPlasmin for thrombolytic therapy

Wang, Y and Zhang, Y and Lin, JJ and Hallock, SJ and Yu, H and Shao, H and Yan, J and Huang, B and Zhang, XC and Cao, W and Xu, X and Lin, X (2013) Purification and characterization of mutant miniPlasmin for thrombolytic therapy. Thrombosis Journal.

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 K<sup>698</sup> 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 K<sup>698</sup> position to develop a mutant form of mPlasmin for thrombolytic therapy.Methods: We changed K<sup>698</sup> 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 K<sup>698</sup>, 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. 2013 Lin et al; licensee BioMed Central Ltd

Item Type: Article
Additional Information: pubid: 185 nvp_institute: NIBR contributor_address: (Lin, Cao, Xu, Lin) Genecopoeia Inc., 9620 Medical Center Drive #101, Rockville, MD 20850, United States (Lin, Zhang, Huang, Lin) Guangzhou FulenGen Co. Ltd., Guangzhou, China (Wang) Clinical Medical and Pharmaceutical College, China Medical University, Shenyang, China (Lin) Novartis Institutes for Biomedical Research, Emeryville, CA, United States (Hallock) Winston Churchill High School, Potomac, MD, United States (Yu) Department of Cardiology, School of Medicine, Zhejiang University, Hangzhou, China (Yu) South Florida VA Foundation for Research, Miami, FL, United States (Yu, Shao) Vascular Biology Institute, University of Miami, Miller School of Medicine, Miami, FL, United States (Yan) Zhejiang Hospital, Hangzhou, China (Huang, Zhang) Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Date Deposited: 13 Oct 2015 13:12
Last Modified: 13 Oct 2015 13:12
URI: https://oak.novartis.com/id/eprint/22006

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