Move recombinant thrombolytic and antithrombotic staphylokinase variants with an RGD motif at their N-termini

作者：He Jintian^*; Di Jingfang; Xu Ruiguang; Zhao Baohua

来源：Biotechnology and Applied Biochemistry, 2008, 50: 17-23.

DOI：10.1042/BA20070191

• antiplatelet aggregation
• Arg-Gly-Asp (RGD) motif
• N-terminal methionine excision
• plasminogen
• staphylokinase
• thrombotic disorders

To develop a more potent thrombolytic agent, four Sak (staphylokinase) variants were constructed, in which RGD (Arg-Gly-Asp) sequences are introduced into diferent sites of the N-terminus of Sak. These variants were successfully expressed in Escherichia coli DHS alpha as soluble cytoplasmic proteins in a 5-litre fermentor and accounted for more than 40% of the total cellular protein. The expressed proteins were subsequently purified, employing a similar three-step chromatographic purification process. SDS/PAGE and HPLC-MS analyses indicated that the purified proteins were almost completely homogeneous, the purity of the variants exceeding 95%. Further investigations into the properties of the Sak variants showed that mutations at the N-terminus significantly affected N-terminal methionine excision, and serine residues at the N-terminus of Sak appeared to play an important role in the process. Kinetic analysis of r-Sak (recombinant Sak) and its variants using plasminogen as substrate indicated that the mutations affected the proteolysis. In addition, a significant inhibitory effect of the Sak variants at 2.0 mu M was observed on the ADP-induced aggregation of platelets compared with that of r-Sak, whether N-terminally cleaved or not (P < 0.05). Furthermore, the inhibitory activity of Sak variants after N-terminal proteolysis was higher than that of native Sak variants.

• 出版日期2008-5
• 单位河北师范大学