Urokinase separation from cell culture broth of a human kidney cell line.
A single step ion-exchange chromatography on a sulfo-propyl (SP)- Sepharose column was performed to separate both the high molecular weight (HMW)- and low molecular weight (LMW)- forms of enzymatically active urokinase type plasminogen activator from human kidney (HT1080) cell culture media. The level of urokinase secreted by the cell line reached to about 145 Plough units/ml culture broth within 48 h of cultivation. The conditioned cell culture media was applied directly to the column without any prior concentration steps. Polyacrylamide gel electrophoresis of the column eluates in the presence of sodium dodecyl sulphate showed that the cell line secretes three forms of two-chain high molecular weight (HMW) urokinase of molecular weights (M(r)) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M(r) 22,000 and 20,000; proteolytic cleavage products of HMW, were also found. The HMW and LMW forms had intrinsic plasminogen dependent proteolytic activity as judged by zymographic analysis. The specific activity of the pooled peak fractions increased (approximately 93-fold) to values as high as 1481 Plough units/ mg protein. Both HMW as well as LMW forms were obtained in significantly high yields.
Bansal
Vibha
V
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz-Khas, New Delhi-110016, INDIA.
Roychoudhury
Pradip K
PK
Kumar
Ashok
A
D002460:Cell Line; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D008970:Molecular Weight; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000302:isolation & purification*; D014568:Urokinase-Type Plasminogen Activator
D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator