Novel recombinant thrombolytic and antithrombotic staphylokinase variants with an RGD motif at their N-termini.
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 DH5alpha 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 muM 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.
He
Jintian
J
College of Life Science, Hebei Normal University, 113 Yuhua East Road, Shijiazhuang City, Hebei Province 050016, People's Republic of China. he_jintian@yahoo.com
Di
Jingfang
J
Xu
Ruiguang
R
Zhao
Baohua
B
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D008715:Methionine / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics; D009842:Oligopeptides / Q000032:analysis*; D010958:Plasminogen / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D013211:Staphylococcus aureus / Q000201:enzymology
D005343:Fibrinolytic Agents; D009842:Oligopeptides; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid; D010958:Plasminogen; D008715:Methionine; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus