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期刊论文
Comparing expression of different forms of human DNA topoisomerase I in Pichia pastoris
Enzyme and Microbial Technology xxx(2003)xxx-xxx,-0001,():
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 (hTopoI△581) 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-hTopoI△ (out) was best suited to express and secrete the highly active hTopoI△ 581 with part of them glycosylated (the enzyme activity of the supernatant reached 3.4×10 8Ul-1). SMD-hTopoI (out) could produce and secrete the full length form of hTopoI without glycosylation (the enzyme activity of the supernatant reached 4.3×10 7Ul-1). The yield of the recombinant hTopoI reached 11mgl-1, achieving 9.7% of the total protein in the culture supernatant, while that of the recombinant hTopoI△ 581 reached 58mgl-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.
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