Expression and characterization of recombinant human micro-plasminogen

Abstract

Micro-plasminogen (microPlg) gene amplified from human liver cells by reverse transcription PCR was inserted into expression vector pET-28a (pET-28a/microPlg) and transformed into E. coli strain BL21(DE3). Recombinant human micro-plasminogen (rh-microPlg) was over-expressed as inclusion bodies when induced with IPTG. After renaturation and purification, 16 mg rh-microPlg/l was obtained with a homogeneity of 95% (w/w). Pro-urokinase (proUK)-induced rh-microPlg activation was significantly faster than when Glu-plasminogen was the substrate. The catalytic efficiency of urokinase (UK) activation of rh-microPlg was twice that of Glu-plasminogen. While recombinant human micro-plasmin (rh-microPlm) and Lys-plasmin had a similar amidolytic activity against a small substrate, D-valyl-L-leucyllysine-p-nitroaniline dihydrochloride, Lys-plasmin activated proUK with a catalytic efficiency about fourfold greater than did rh-microPlm. These results suggested that the kringle 1-5 domain of plasminogen and plasmin may modify both UK activation of plasminogen and plasmin activation of proUK, respectively.

Binding of Tissue-type Plasminogen Activator to the Glucose-regulated Protein 78 (GRP78) Modulates Plasminogen Activation and Promotes Human Neuroblastoma Cell Proliferation in Vitro

“…Human VDAC clones (Genecopeia, Germantown, MD) were expressed in Escherichia coli and purified as previously described (20). Recombinant human microplasminogen (Genecopeia) was expressed in E. coli and purified from clones as previously described (21). Recombinant murine GRP78 and the COOH-terminal domain of GRP78 containing amino acids 516 -636 (Lys 516 -Gly 636 ), a kind gift from Dr. Sylvie Y.…”

“…The velocity of S-2288 hydrolysis was calculated as the increase of absorbance at 405 nm using a Molecular Devices SPECTRAmax kinetic plate reader. The molar extinc-tion coefficient (⑀) of p-nitroanilide at 405 nm is 10,000 M Ϫ1 cm Ϫ1 (21). The kinetic parameters (K m ,V max , and k cat ) were calculated using the GraphPad Prism 6 program from GraphPad Software, Inc.…”

“…Determination of Pg Activation Rate-Coupled assays were used to evaluate the initial rate of Glu-Pg activation by t-PA by monitoring the amidolytic activity of the generated plasmin (Pm) (21). Glu-Pg (100 nM) was incubated in 96-well microtiter plates at 37°C in 20 mM HEPES, pH 7.4, in a total volume of 200 l with the plasmin substrate S-2251 (0.3 mM).…”

The evolution of recombinant thrombolytics: Current status and future directions

“…144 The molecule has been recombinantly expressed in E. coli, Pichia pastoris, and insect cells. 149,150,151 It showed 100 times lesser inhibition by a2-antiplasmin than plasmin due to the absence of the lysine binding sites in micro-plasmin. 150 It does not have any kringle domain and thus lacks fibrin affinity.…”

A novel human microplasmin fold: new perspective to thrombosis treatment

“…Sequence analysis has shown that the NH 2 terminal portion of the plasminogen molecule, which becomes the heavy (A) chain after activation, contains homologous domains referred to as 'kringles' [3]. It appears that the kringles contain lysine-binding sites which are involved in the interaction of plasmin with fibrin and a-2-antiplasmin [4][5][6][7][8]. The fibrinolytic activity in human plasma was subsequently shown to depend on activation of a plasminogen as precursor.…”