Preparation of a recombinant tumor-targeting ribosome inactivating protein luffin-α-NGR and evaluation of its antitumor activity.
- Author:
Zheyue ZHOU
1
;
Xinyi JIANG
1
;
Hongrui ZHANG
1
;
Zhiguang HUANG
1
;
Rui ZOU
1
;
Qiuwen LOU
1
;
Yu WANG
1
;
Zhenhong ZHU
1
Author Information
- Publication Type:Journal Article
- Keywords: anti-tumor; loofah; luffin-α; ribosome inactivating protein; targeting peptide
- MeSH: Electrophoresis, Polyacrylamide Gel; Escherichia coli/metabolism*; Plasmids; Recombinant Proteins/pharmacology*; Saporins/metabolism*
- From: Chinese Journal of Biotechnology 2022;38(3):1138-1148
- CountryChina
- Language:Chinese
- Abstract: Loofah seeds ribosome inactivating protein luffin-α was fused with a tumor-targeting peptide NGR to create a recombinant protein, and its inhibitory activity on tumor cells and angiogenesis were assessed. luffin-α-NGR fusion gene was obtained by PCR amplification. The fusion gene was ligated with pGEX-6p-1 vector to create a recombinant plasmid pGEX-6p-1/luffin-α-NGR. The plasmid was transformed into E. coli BL21, and the target protein was isolated and purified by GST affinity chromatography. The luffin-α-NGR fusion gene with a full length of 849 bp was successfully obtained, and the optimal soluble expression of the target protein was achieved under the conditions of 16 ℃, 0.5 mmol/L IPTG after 16 h induction. SDS-PAGE and Western blotting confirmed the recombinant protein has an expected molecular weight of 56.6 kDa. Subsequently, the recombinant protein was de-tagged by precision protease digestion. The inhibitory effects of the recombinant protein on liver tumor cells HepG2 and breast cancer cells MDA-MB-231 were significantly stronger than that of luffin-α. The Transwell and CAM experiment proved that the recombinant protein luffin-α-NGR also had a significant inhibitory effect on tumor cells migration and neovascularization. The inhibitory activity on tumor cells and angiogenesis of the recombinant luffin-α-NGR protein lays a foundation for the development of subsequent recombinant tumor-targeting drugs.
