Construction and expression of antibody targeted plasminogen activator

It has been known that antibody-mediated plasminogen activator wilt be much more specific than its parent molecular. To get a cheaper and more effecti ve medicine for thrombolytic therapy, we used SZ51, a GMP140 specific monoc lonal antibody, and a truncated single-chain urokinase to construct a novel targeted plasminogen activator. PCR was used to amplify the region of VL a nd VH chains from Fab of SZ51, GMP140 specific monoclonal antibody, and scu -PA-32KD(leu144-leu411) from urokinase gene, respectively. Through suitable linker and appropriate restriction sites, these fragments were joined toge ther and inserted into the expression vector, pET-5a, via NdeI site. The re combinant protein was expressed in BL21 (DE3) plyS, a kind of E. coli. It w as shown in Western-blotting and ELISA that the protein could interact with the multiple cloned antibody of urokinase. After partial purification: dia lysis, Sephadex G-100, dialysis and Phenyl-Sepharose fast flow, the product had a strong fibrinolytic activity through activating plasminogen on fibri n plate. The specific activity was about 47,000 IU/mg, corresponding to 80, 000 IU/mg for the part of rscu-PA-32k, and the activity could be inhibited specifically by urokinase specific antibody. Activation of plasminogen by t he chimera followed Michaelis-Menten kinetics, and the K-m was 1.08 uM. (C) 2000 Elsevier Science Inc. All rights reserved.