Purification and characterization of mutant miniPlasmin for thrombolytic therapy
1 Genecopoeia Inc, 9620 Medical Center Drive #101, 20850, Rockville, MD, USA
2 Guangzhou FulenGen Co., Ltd, Guangzhou, China
3 Clinical Medical and Pharmaceutical College, China Medical University, Shenyang, China
4 Novartis Institutes for Biomedical Research, Emeryville, CA, USA
5 Winston Churchill High School, Potomac, MD, USA
6 Department of Cardiology, School of Medicine, Zhejiang University, Hangzhou, China
7 South Florida VA Foundation for Research, Miami, FL, USA
8 Vascular Biology Institute, University of Miami, Miller School of Medicine, Miami, FL, USA
9 Zhejiang Hospital, Hangzhou, China
10 Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Thrombosis Journal 2013, 11:2 doi:10.1186/1477-9560-11-2Published: 30 January 2013
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.
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.
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.
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.