Subcloning of human topoisomerasa I in E. coli.

CHIALVA CS; ZÁRATE JM; LAPADULA WJ; PUNGITORE CR; JURI AYUB M

Mendoza

Congreso; Sociedad Biología de Cuyo; 2010

Sociedad de Biología de Cuyo

Human topoisomerase I (hTopo I) is a 91 kDa protein of 765 aa bearing three structural domains. The catalytic activity of the enzyme resides in both core and C-terminal domain containing the active Tyr 723. It has previously been described a yeast model for the study of eukaryotic Topo I. Therefore, in the present work we intended to develop an E. coli system for the expression of hTopo I. Secuences encoding the C-terminal active domains were obtained from the full length cDNA using the polymerase chain reaction and cloned into the pTrcHisTOPO expression vector. Recombinant vectors were transformed in E. coli BL21 (DE3) pLysS. Induction of protein expression with IPTG led consistently to the expression of truncated proteins, strongly suggesting that the enzyme was toxic for bacterial cells. In light of these results, other experimental approaches will be needed for obtaining recombinant hTopo I, such as periplasmic expression using exportation signals, or vectors based in T7 RNA polymerase in order to reduce basal expression.