Asn12 and Asn278: Critical Residues for In Vitro Biological Activity of Reteplase
Reteplase (rPA) is a thrombolytic agent used for the treatment of acute myocardial infarction. We studied the expression of rPA and its selected asparagine mutants after integration into the Pichia genome. Though methanol induction of the native and the rPA mutants showed similar expression levels (...
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| Format: | Article |
| Language: | English |
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Wiley
2010-01-01
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| Series: | Advances in Hematology |
| Online Access: | http://dx.doi.org/10.1155/2010/172484 |
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| author | Naganath Mandi Kalyana R. Sundaram Sunil K. Tandra Suman Bandyopadhyay Sriram Padmanabhan |
| author_facet | Naganath Mandi Kalyana R. Sundaram Sunil K. Tandra Suman Bandyopadhyay Sriram Padmanabhan |
| author_sort | Naganath Mandi |
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| description | Reteplase (rPA) is a thrombolytic agent used for the treatment of acute myocardial infarction. We studied the expression of rPA and its selected asparagine mutants after integration into the Pichia genome. Though methanol induction of the native and the rPA mutants showed similar expression levels (~200–250 mg/L), the mutants displayed significant loss of protease activity. Strikingly, the clot lysis activities of these mutants were considerably different. While mutation of Asn12 (N12P) of the Kringle 2 domain showed delayed clot lysis activity (𝑡1/2=38 min) compared to the native rPA (𝑡1/2=33 min), a faster rate of clot lysis (𝑡1/2=27 min) was observed when the Asn278 (N278S) of the serine protease domain was mutated. Interestingly, the slowest clot lysis activity (𝑡1/2=49 min) demonstrated by the double mutant (N12P, N278S) suggests the dominant role of Asn12 in regulating the fibrinolytic activity of rPA. The results presented in this paper indicate that the fibrinolytic and the proteolytic activities of rPA are independent of each other. |
| format | Article |
| id | doaj-art-2f3badf3dacb454aa59e74766e82a804 |
| institution | Kabale University |
| issn | 1687-9104 1687-9112 |
| language | English |
| publishDate | 2010-01-01 |
| publisher | Wiley |
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| series | Advances in Hematology |
| spelling | doaj-art-2f3badf3dacb454aa59e74766e82a8042025-08-20T03:55:16ZengWileyAdvances in Hematology1687-91041687-91122010-01-01201010.1155/2010/172484172484Asn12 and Asn278: Critical Residues for In Vitro Biological Activity of ReteplaseNaganath Mandi0Kalyana R. Sundaram1Sunil K. Tandra2Suman Bandyopadhyay3Sriram Padmanabhan4Clone Development Team, Lupin Limited, Biotechnology R & D, Gat #1156, Mulshi Taluka, Ghotawade Village, Pune 411042, IndiaAnalytical Development Team, Lupin Limited, Biotechnology R & D, Gat #1156, Mulshi Taluka, Ghotawade Village, Pune 411042, IndiaMammalian Bioassay Team, Lupin Limited, Biotechnology R & D, Gat #1156, Mulshi Taluka, Ghotawade Village, Pune 411042, IndiaMammalian Bioassay Team, Lupin Limited, Biotechnology R & D, Gat #1156, Mulshi Taluka, Ghotawade Village, Pune 411042, IndiaLupin Limited, Biotechnology R & D, Gat #1156, Mulshi Taluka, Ghotawade Village, Pune 411042, IndiaReteplase (rPA) is a thrombolytic agent used for the treatment of acute myocardial infarction. We studied the expression of rPA and its selected asparagine mutants after integration into the Pichia genome. Though methanol induction of the native and the rPA mutants showed similar expression levels (~200–250 mg/L), the mutants displayed significant loss of protease activity. Strikingly, the clot lysis activities of these mutants were considerably different. While mutation of Asn12 (N12P) of the Kringle 2 domain showed delayed clot lysis activity (𝑡1/2=38 min) compared to the native rPA (𝑡1/2=33 min), a faster rate of clot lysis (𝑡1/2=27 min) was observed when the Asn278 (N278S) of the serine protease domain was mutated. Interestingly, the slowest clot lysis activity (𝑡1/2=49 min) demonstrated by the double mutant (N12P, N278S) suggests the dominant role of Asn12 in regulating the fibrinolytic activity of rPA. The results presented in this paper indicate that the fibrinolytic and the proteolytic activities of rPA are independent of each other.http://dx.doi.org/10.1155/2010/172484 |
| spellingShingle | Naganath Mandi Kalyana R. Sundaram Sunil K. Tandra Suman Bandyopadhyay Sriram Padmanabhan Asn12 and Asn278: Critical Residues for In Vitro Biological Activity of Reteplase Advances in Hematology |
| title | Asn12 and Asn278: Critical Residues for In Vitro Biological Activity of Reteplase |
| title_full | Asn12 and Asn278: Critical Residues for In Vitro Biological Activity of Reteplase |
| title_fullStr | Asn12 and Asn278: Critical Residues for In Vitro Biological Activity of Reteplase |
| title_full_unstemmed | Asn12 and Asn278: Critical Residues for In Vitro Biological Activity of Reteplase |
| title_short | Asn12 and Asn278: Critical Residues for In Vitro Biological Activity of Reteplase |
| title_sort | asn12 and asn278 critical residues for in vitro biological activity of reteplase |
| url | http://dx.doi.org/10.1155/2010/172484 |
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