Multiple gene copy number increases total protein expression and enzyme activity of DNA topoisomerase I in Pichia pastoris

Abstract

The present study aimed to employ an in vivo strategy for the establishment of multi-copy gene constructs of human DNA topoisomerase I (hTopI) using the pPIC3.5K vector in Pichia. The clones with multi-copy inserts (His+ transformants) that were able to survive in the highest concentration of Geneticin® were found to express the highest expression level of total protein and exhibited the highest target enzyme activity. The highest level of total protein found was 1.76 mg/ml in GS115-pPIC3.5K-hTop1, which was resistant to 1.00 mg/ml Geneticin at 48 h of incubation. The highest enzyme activity of hTopI was also observed in the culture expressed by GS115-pPIC3.5K-hTopI, which was resistant to 1.00 mg/ml Geneticin® (19.7x104 Ul/OD600). On the whole, the present study provides information regarding the production of target protein from recombinant Pichia using only a shaker flask system, which can be further developed as an in-house resource for screening potential anticancer agents.