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A scalable and reproducible preparation for the antitumor protein TLC, a human-derived telomerase inhibitor
Protein Expression and Purification ( IF 1.6 ) Pub Date : 2021-07-17 , DOI: 10.1016/j.pep.2021.105942
Guangming Chen 1 , Xiaoying Zhang 1 , Wei He 2 , Mingzhe Shao 3 , Qianli Gao 4 , Linlin Li 4 , Mujun Zhao 5 , Hongchang Zhou 1
Affiliation  

Telomerase, which is overexpressed in approximately 90% of liver cancer cells, is an ideal target for anti-liver cancer therapy. LPTS, a putative liver tumor suppressor, is the only human-derived protein that can bind telomerase directly and inhibit the extension of telomere activity. Our previous studies demonstrated that TAT-LPTS-LC (TLC), a recombinant protein fused by the C-terminal 133–328 fragment of LPTS and TAT peptides, could be delivered into cells to inhibit telomerase-positive hepatoma cell growth in vitro and in vivo with very low toxicity. In the present study, E. coli strains which expressed TLC in abundance were screened and cultured in a laboratory bioreactor. A reproducible protein separation process was built, and this process was suitable for industrial amplification. The yields of TLC protein were up to 184 mg in one batch with a purity of approximately 95%. The purified TLC protein had a similar inhibitory effect on telomerase activity in vitro compared with those purified by Ni-affinity chromatography. Furthermore, TLC protein could be delivered into the cell nucleus to increase the doubling time of the cell and suppress cell growth in telomerase-positive liver cancer cell lines. Cell growth inhibition was negatively correlated with telomere length, suggesting that TLC is a highly targeted telomerase-telomere anticancer agent. These results will contribute to future preclinical studies of the TLC protein.



中文翻译:

一种可扩展且可重复的抗肿瘤蛋白 TLC 制剂,一种人源端粒酶抑制剂

端粒酶在大约 90% 的肝癌细胞中过度表达,是抗肝癌治疗的理想靶点。LPTS 是一种推定的肝脏肿瘤抑制因子,是唯一一种可以直接结合端粒酶并抑制端粒活性延伸的人源蛋白。我们之前的研究表明,TAT-LPTS-LC (TLC) 是一种重组蛋白,由 LPTS 的 C 末端 133-328 片段和 TAT 肽融合而成,可以在体外和体内递送到细胞中以抑制端粒酶阳性肝癌细胞的生长。体内毒性极低。在本研究中,大肠杆菌在实验室生物反应器中筛选和培养大量表达 TLC 的菌株。建立了可重现的蛋白质分离工艺,该工艺适用于工业放大。一批TLC蛋白的产量高达184 mg,纯度约为95%。纯化的 TLC 蛋白在体外对端粒酶活性的抑制作用与 Ni 亲和层析纯化的相比具有相似的抑制作用。此外,TLC蛋白可以被递送到细胞核中以增加细胞的倍增时间并抑制端粒酶阳性肝癌细胞系中的细胞生长。细胞生长抑制与端粒长度呈负相关,表明 TLC 是一种高度靶向的端粒酶-端粒抗癌剂。

更新日期:2021-07-20
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