Urokinase Separation from Cell Culture Broth of a Human Kidney Cell Line
<p>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...
| Main Author: | |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Ivyspring International Publisher
2007-01-01
|
| Series: | International Journal of Biological Sciences |
| Online Access: | http://www.biolsci.org/v03p0064.htm |
| _version_ | 1819208486804783104 |
|---|---|
| author | Vibha Bansal, Pradip K. Roychoudhury, Ashok Kumar |
| author_facet | Vibha Bansal, Pradip K. Roychoudhury, Ashok Kumar |
| author_sort | Vibha Bansal, Pradip K. Roychoudhury, Ashok Kumar |
| collection | DOAJ |
| description | <p>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<sub>r</sub>) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M<sub>r</sub> 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.</p> |
| first_indexed | 2024-12-23T05:40:10Z |
| format | Article |
| id | doaj.art-237c9eed556d4daaa9929e8c4818a070 |
| institution | Directory Open Access Journal |
| issn | 1449-2288 |
| language | English |
| last_indexed | 2024-12-23T05:40:10Z |
| publishDate | 2007-01-01 |
| publisher | Ivyspring International Publisher |
| record_format | Article |
| series | International Journal of Biological Sciences |
| spelling | doaj.art-237c9eed556d4daaa9929e8c4818a0702022-12-21T17:58:13ZengIvyspring International PublisherInternational Journal of Biological Sciences1449-22882007-01-01316470Urokinase Separation from Cell Culture Broth of a Human Kidney Cell LineVibha Bansal, Pradip K. Roychoudhury, Ashok Kumar<p>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<sub>r</sub>) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M<sub>r</sub> 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.</p>http://www.biolsci.org/v03p0064.htm |
| spellingShingle | Vibha Bansal, Pradip K. Roychoudhury, Ashok Kumar Urokinase Separation from Cell Culture Broth of a Human Kidney Cell Line International Journal of Biological Sciences |
| title | Urokinase Separation from Cell Culture Broth of a Human Kidney Cell Line |
| title_full | Urokinase Separation from Cell Culture Broth of a Human Kidney Cell Line |
| title_fullStr | Urokinase Separation from Cell Culture Broth of a Human Kidney Cell Line |
| title_full_unstemmed | Urokinase Separation from Cell Culture Broth of a Human Kidney Cell Line |
| title_short | Urokinase Separation from Cell Culture Broth of a Human Kidney Cell Line |
| title_sort | urokinase separation from cell culture broth of a human kidney cell line |
| url | http://www.biolsci.org/v03p0064.htm |
| work_keys_str_mv | AT vibhabansalpradipkroychoudhuryashokkumar urokinaseseparationfromcellculturebrothofahumankidneycellline |