Single-chain urokinase-type plasminogen activator (scu-PA) purification by immuno-affinity chromatography.
- Author:
Li-Hua GAO
1
;
Xian-Wen HU
;
Qing-Fa WU
;
Cheng-Zu XIAO
;
Zhao-Ping XU
;
Zheng-Guang ZHANG
Author Information
1. Institute of Biotechnology, Beijing 100071, China. Lihuagao@hotmail.com
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies, Monoclonal;
immunology;
isolation & purification;
Chromatography, Affinity;
Enzyme-Linked Immunosorbent Assay;
Mice;
Mice, Inbred BALB C;
Recombinant Proteins;
immunology;
isolation & purification;
Urokinase-Type Plasminogen Activator;
immunology;
isolation & purification
- From:
Chinese Journal of Biotechnology
2002;18(3):356-359
- CountryChina
- Language:Chinese
-
Abstract:
The only difference of primary structure between single-chain prourokinase (pro-UK or scu-PA) and two-chain urokinase (UK or tcu-PA) is the cleavage of a single peptide bond (Lys158-Ile159) and transform scu-PA into its active two-chain form. A 13-peptide (Thr-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly-Glu-Cys), which spans the cleavage peptide bond, was synthesized and linked to KLH (Keyhole limpet hemocyanin). The Balb/c mice were immunized by the conjugated protein with proper adjuvant. According to the Kohler and Milstein's methods, a hybridoma cell line G7 secreting monoclonal antibody specific for scu-PA was obtained. The anti-scu-PA McAb, purified from the supernatant of porous microcarrier hybridoma cell culture, was conjugated to CNBr-activated Sepharose 4B to prepare an immuno-affinity chromatography column. The u-PA was purified only by this affinity column from the supernatant of cultivating the u-PA-producing recombinant CHO cell, the u-PA recovery ratio is 90.4%, the purification factor was about 50, with the specific activity of 1.2 x 10(5) IU/mg, the scu-PA ratio in the u-PA product was 96.3%. Compared to immuno-affinity chromatography, the 3-step process for purifying u-PA (cation-exchange column, gel filtration column and benzamidine affinity column) has a u-PA recovery ratio of about 65%, with a specific activity of 1.0 x 10(5) IU/mg, and an scu-PA ratio of about 90%. These results showed that immuno-affinity chromatography is simple to recover u-PA and effective to separate scu-PA from tcu-PA.
