RESUMO
Abstract The effect of the intracellular microenvironment in the presence of an oxygen vector during expression of a fusion protein in Escherichia coli was studied. Three organic solutions at different concentration were chosen as oxygen vectors for fumarase expression. The addition of n-dodecane did not induce a significant change in the expression of fumarase, while the activity of fumarase increased significantly to 124% at 2.5% n-dodecane added after 9 h induction. The concentration of ATP increased sharply during the first 6 h of induction, to a value 7600% higher than that in the absence of an oxygen-vector. NAD/NADH and NADP/NADPH ratios were positively correlated with fumarase activity. n-Dodecane can be used to increase the concentration of ATP and change the energy metabolic pathway, providing sufficient energy for fumarase folding.
Assuntos
Oxigênio/metabolismo , Expressão Gênica , Alcanos/metabolismo , Escherichia coli/genética , Fumarato Hidratase/metabolismo , Oxigênio/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Dobramento de Proteína , Alcanos/química , Escherichia coli/metabolismo , Fumarato Hidratase/genética , Fumarato Hidratase/química , NADP/metabolismo , NADP/químicaRESUMO
The NADPH-dependent reduction activities of two paralogous pig AKR1C1s with and without 19 additional amino acid residues in C-terminus were evaluated against steroid hormones including 5a-dihydrotestosterone, testosterone, progesterone, androstenedione and 5a-androstane-3,17-dione, which act as substrates of the AKR1C1s. Among the hormones, the AKR1C1s exhibited the highest activity against 5a-dihydrotestosterone and the lowest activity against testosterone and progesterone. Furthermore, the AKR1C1s showed the largest differential activities against 5a-dihydrotestosterone, but no such change of activities was found against progestrone and testosterone. These results suggest that the C-terminal region of AKR1C1 plays an important effect in the reduction activities of pig AKR1C1. Thus, the differential activities of two AKR1C1 paralogs observed in the present study provide important insights in understanding the molecular evolution.