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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. • A scalable and reproducible preparation process was established for anti-tumor recombinant protein TLC, which was a human-derived telomerase inhibitor protein, from the E. coli BL21 (DE3) cells with high purity. • The purified TLC protein had same activities as TLC-His protein with a C-terminal 6X His tag purified by Ni-affinity chromatography reported earlier. • The TLC could inhibit the growth of 8 kinds of telomerase-positive liver cancer cell lines, but not the telomerase-negative normal liver cells. • The inhibition rate for liver cancer cells was negatively correlated with telomere length in the cells, indicating that TLC has high specificity and low toxicity for anti-liver cancer therapy. [ABSTRACT FROM AUTHOR]