Sequential and simultaneous statistical optimization by dynamic design of experiment for peptide overexpression in recombinant Escherichia coli

Abstract

The production of recombinant anti-HIV peptide, T-20, in Escherichia coli was optimized by statistical experimental designs (successive designs with multifators) such as 2^4–1 fractional factorial, 2³ full factorial, and 2² rotational central composite design in order. The effects of media compositions (glucose, NPK sources, MgSO₄, and trace elements), induction level, induction timing (optical density at induction process), and induction duration (culture time after induction) on T-20 production were studied by using a statistical response surface method. A series of iterative experimental designs was employed to determine optimal fermentation conditions (media and process factors). Optimal ranges characterized by %T-20 (proportion of pepttide to the total cell protein) were observed, narrowed down, and further investigated to determine the optimal combination of culture conditions, which was as follows: 9, 6, 10, and 1 mL of glucose, NPK sources, MgSO₄, and trace elements, respectively, in a total of 100 mL of medium inducted at an OD of 0.55–0.75 with 0.7 mM isopropyl-β-d-thiogalactopyranoside in an induction duration of 4 h. Under these conditions, up to 14% of T-20 was obtained. This statistical optimization allowed, the production of T-20 to be increased more than twofold (from 6 to 14%) within, a shorter induction duration (from 6 to 4 h) at the shake-flask scale.