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.
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
Jintian
J
He
Jingfang
J
Di
Ruiguang
R
Xu
Baohua
B
Zhao
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