Purification and characterization of a staphylokinase variant, K35R.
DGR [staphylokinase (Sak) variant K35R, in which lysine (K) at position 35 is replaced by arginine (R)], a recombinant mutant of the Staphylococcus aureus enzyme, is a promising drug for thrombotic disorders. In the present work, DGR was successfully overexpressed by the plasmid JF1125[pST-DGR] as a soluble cytoplasmic protein in a 30-litre fermentor that accounted for more than 50% of the total cellular protein. The expressed DGR was subsequently purified by using a simple three-step chromatographic purification process developed at a pilot scale. The clearance of host-cell-protein contaminants in the protein purification process was confirmed by SDS/PAGE and Western blotting, using rabbit antisera raised against Escherichia coli JF1125 cell proteins. SDS/PAGE, isoelectric focusing and HPLC-MS analysis indicated that the purified DGR is almost completely homogeneous. The purification process resulted in greater-than-98% pure DGR and yielded up to 25.0 mg/g wet weight of cells. The effect of pH and temperature on the stability of DGR was investigated further. The results showed that DGR was highly stable at neutral pH and more stable than two other wild-type Saks, SakSTAR and Sak42D, when submitted to high temperatures.
He
Jintian
J
College of Life Science, Hebei Normal University, Shijiazhuang City, Hebei Province, People's Republic of China. he_juntian@yahoo.com
Wang
Gaizhen
G
Li
Guoping
G
Zhao
Baohua
B
Mo
Wei
W
Song
Houyan
H
D019149:Bioreactors; D002852:Chromatography, Ion Exchange / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007218:Industrial Microbiology / Q000379:methods*; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000187:drug effects / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000302:isolation & purification; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000494:pharmacology
D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus