and : Critical Residues for In Vitro Biological Activity of Reteplase

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 Asn12 (N12P) of the Kringle 2 domain showed delayed clot lysis activity (t 1/2 = 38 min) compared to the native rPA (t 1/2 = 33 min), a faster rate of clot lysis (t 1/2 = 27 min) was observed when the Asn278 (N278S) of the serine protease domain was mutated. Interestingly, the slowest clot lysis activity (t 1/2 = 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.

Molecular cloning of Reteplase and its expression in E. coli using tac promoter

“…In this study we followed the aim of cloning and expression of reteplase in E. coli using tac promoter. In comparison to t-PA, reteplase is a single chain deletion mutant of t-PA[ 2 4 ] containing 355 amino acid residues with molecular mass of 39 kDa. The Kringle II and Protease domains of t-PA is kept in reteplase and it retains enzymatic activity that is comparable to the native t-PA in vitro .…”

“…[ 2 7 ] It is used for treating occlusive disorders of the lungs, heart, and brain. [ 3 4 5 6 ] t-PA has been expressed in different systems such as bacteria, fungi, insect, yeasts, Chinese Hamster Ovary (CHO) and mammalian cell lines. [ 4 8 9 16 17 ] A problem occurring when mammalian cell lines are used is glycosylation of the product which leads to the rapid clearance of this drug.…”

“…[ 3 4 5 6 ] t-PA has been expressed in different systems such as bacteria, fungi, insect, yeasts, Chinese Hamster Ovary (CHO) and mammalian cell lines. [ 4 8 9 16 17 ] A problem occurring when mammalian cell lines are used is glycosylation of the product which leads to the rapid clearance of this drug. [ 1 ] Also because of wrong folding, low secretion to the culture media and high level of glycosylation, yeasts like Saccharomyces cervisiae and insect cells are unsuitable expression systems.…”

“…[ 1 2 ] It is used for the treatment of occlusive disorders, especially myocardial infarction which is responsible for more than 50% of cardiovascular events deaths. [ 3 4 5 6 ] One of the important clinical advantages of t-PA is that it produces no side effects such as systemic hemorrhage and fibrinogen depletion and therefore many researchers are working towards the reduction of the cost of its production. [ 1 2 7 ] In comparison to tissue plasminogen activator (t-PA), reteplase is nonglycosylated and has longer half life.…”

“…Reteplase is expressed in various bacterial, fungal, mammalian and insect systems. [ 4 8 9 ] However due to the variability of expression levels of r-PA in these systems, problems of protein instability and downstream processing, each expression system differs for its capabilities for the production of reteplase. Although the expression of r-PA in bacterial systems like E. coli has been investigated by some researchers, various cloning and expression plasmids and conditions can affect the yield and production characteristics of reteplase.…”