Comparing expression of different forms of human DNA topoisomerase I in Pichia pastoris

In order to rapidly screen new inhibitors of human DNA topoisomerase I in vitro, the enzyme has been successfully overexpressed in the full-length form (hTopoI) and N-terminal 184 amino acids truncated form (hTopoIΔ581) in Pichia pastoris by the secretory and intracellular styles. The results showed that the recombinant enzyme containing nuclear localization signals obviously decreased its expression level. If the enzyme was fused to α-factor secretory signal or the AKL triplet amino acids (a C-terminal peroxisomal-targeting signal), its output could be highly improved. The recombinant strain SMD-hTopoIΔ(out) was best suited to express and secrete the highly active hTopoIΔ581 with part of them glycosylated (the enzyme activity of the supernatant reached 3.4×108 U l−1). SMD-hTopoI(out) could produce and secrete the full length form of hTopoI without glycosylation (the enzyme activity of the supernatant reached 4.3×107 U l−1). The yield of the recombinant hTopoI reached 11 mg l−1, achieving 9.7% of the total protein in the culture supernatant, while that of the recombinant hTopoIΔ581 reached 58 mg l−1, achieving 47.3% of the total protein in the supernatant. Both forms of the recombinant enzyme exhibited the same properties as the natural hTopoI.