Construction and Characterization of Novel Staphylokinase Variants with Antiplatelet Aggregation Activity and Reduced Immunogenecity

Hua-Bo SU, Yu-Gao
ZHANG, Jin-Tian HE, Wei MO, Yan-Ling ZHANG, Xian-Mei TAO, and Hou-Yan SONG*
( Department of Molecular Genetics, Shanghai Medical School, Fudan University,
Key Laboratory of Molecular Medicine, Ministry of Education,
Shanghai
200032, China )

Abstract To develop target
thrombolytic agents with fibrinolytic activity, antiplatelet aggregation activity
and reduced immunogenicity, two staphylokinase variants containing Arg-Gly-Asp
(RGD) motif were constructed. Gene expression was induced in E. coli JF1125
and the variants, designated DGR and RL1, were
purified with gel filtration and ion-exchange chromatography and the purity
was over 95%. The fibrinolytic activity and kinetic constants of the two variants
were comparable to those of recombinant wild-type staphylokinase. Both the variants
can inhibit the platelet aggregation at a final concentration of 2 μM. The
titers of antibodies against variants were much lower than those against recombinant
staphylokinase in guinea pigs, which indicated that the immunogenicity of the
variants was greatly reduced. These results confirm that it is possible to design
and produce a bifunctional protein that possesses fibrinolytic and antiplatelet
aggregation activities.ed that the recombinant proteins had a significant hypocalcemic
effect on mice sera.

Key words OPG-372; silkworm
baculovirus expression system; hypocalcemic effect

—————–

Received: February 20,2004 Accepted: April 6, 2004
*Corresponding author: Tel，86-21-64033738; E-mail，[email protected]