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Advances in Hematology
Volume 2010, Article ID 172484, 9 pages
http://dx.doi.org/10.1155/2010/172484
Research Article

A s n 1 2 and A s n 2 7 8 : Critical Residues for In Vitro Biological Activity of Reteplase

1Clone Development Team, Lupin Limited, Biotechnology R & D, Gat #1156, Mulshi Taluka, Ghotawade Village, Pune 411042, India
2Analytical Development Team, Lupin Limited, Biotechnology R & D, Gat #1156, Mulshi Taluka, Ghotawade Village, Pune 411042, India
3Mammalian Bioassay Team, Lupin Limited, Biotechnology R & D, Gat #1156, Mulshi Taluka, Ghotawade Village, Pune 411042, India
4Lupin Limited, Biotechnology R & D, Gat #1156, Mulshi Taluka, Ghotawade Village, Pune 411042, India

Received 23 January 2010; Accepted 25 May 2010

Academic Editor: Henny H. Billett

Copyright © 2010 Naganath Mandi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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 A s n 1 2 (N12P) of the Kringle 2 domain showed delayed clot lysis activity ( 𝑡 1 / 2 = 3 8  min) compared to the native rPA ( 𝑡 1 / 2 = 3 3  min), a faster rate of clot lysis ( 𝑡 1 / 2 = 2 7  min) was observed when the A s n 2 7 8 (N278S) of the serine protease domain was mutated. Interestingly, the slowest clot lysis activity ( 𝑡 1 / 2 = 4 9  min) demonstrated by the double mutant (N12P, N278S) suggests the dominant role of A s n 1 2 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.