Optimization of the preparation process for fusion protein Fv-LDP that composes lidamycin apoprotein and single-chain Fv antibody directed against type IV collagenase.
- Author:
Rui-Juan GAO
1
;
Chun-Yan ZHAO
1
;
Dian-Dong LI
1
;
Yong-Su ZHEN
1
Author Information
1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
- Publication Type:Journal Article
- MeSH:
Adenocarcinoma;
metabolism;
pathology;
Aminoglycosides;
chemistry;
metabolism;
Antibiotics, Antineoplastic;
chemistry;
metabolism;
Apoproteins;
chemistry;
metabolism;
Carcinoma, Hepatocellular;
metabolism;
pathology;
Cell Line, Tumor;
Collagenases;
immunology;
Enediynes;
chemistry;
metabolism;
Escherichia coli;
chemistry;
metabolism;
Humans;
Inclusion Bodies;
chemistry;
metabolism;
Liver Neoplasms;
metabolism;
pathology;
Lung Neoplasms;
metabolism;
pathology;
Protein Binding;
Recombinant Fusion Proteins;
chemistry;
metabolism;
Single-Chain Antibodies;
chemistry;
metabolism
- From:
Acta Pharmaceutica Sinica
2013;48(10):1563-1569
- CountryChina
- Language:Chinese
-
Abstract:
This study is to optimize the preparation process of fusion protein Fv-LDP which was expressed in the form of inclusion body and consisted of lidamycin apoprotein LDP and single-chain Fv antibody (scFv) directed against type IV collagenase. The preparation and the dissolution of inclusion body, the immobilized metal affinity chromatography of the target protein and the renaturization by stepwise dialysis were optimized by single-factor analysis or orthogonal design. In addition, the refolded fusion protein Fv-LDP was refined by Sephadex G-75 chromatography followed by fluorescence-activated cell sorter (FACS)-based saturation binding assay to measure its antigen-binding activity. After optimization of the process, the purity of fusion protein Fv-LDP existed in the inclusion body was 63.9% and the corresponding solubility was 95.7%; Under denaturing conditions, the purity of fusion protein Fv-LDP was more than 95% after the purification process. The percentage of monomeric fusion protein Fv-LDP was 60% after the refolding process, while it was further refined to 85% which was 5.6-fold higher than that of the initial refolding condition. The refined fusion protein Fv-LDP could bind to human lung adenocarcinoma PAa cells and human hepatoma BEL-7402 cells with the dissociation constants (Kd) of 0.176 micromol x L(-1) and 0.904 micromol x L(-1), respectively. The preparation process of fusion protein Fv-LDP has been successfully optimized, which provides the experimental basis for the production and future development of fusion protein Fv-LDP, and might serve as a relatively practical system for the preparation of other scFv-based proteins expressed in the form of inclusion body.
