Optimization of recombinant maize CDKA;1 and CycD6;1 production in Escherichia coli by response surface methodology

MÉNDEZ, ANDREA A.E.; PENA, LILIANA B.; CURTO, LUCRECIA M.; SCIORRA, MARCELO D.; ULLOA, RITA M.; GARZA AGUILAR, SARA M.; VÁZQUEZ RAMOS, JORGE M.; GALLEGO, SUSANA M.

PROTEIN EXPRESSION AND PURIFICATION

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2019

1046-5928

The complex formed by the cyclin-dependent kinase A (CDKA) and cyclin D is responsible for the G1-S transition in the plant cell cycle. Maize (Zea mays L) CDKA; 1 and CycD6; 1 were cloned and expressed in E. coli. The present study describes the optimization of both proteins production using a statistical approach known as response surface methodology (RSM). The experimental design took into account the effects of four variables: optical density of the culture (OD600) before induction, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration, post-induction temperature, and post-induction time. For each protein, a 24−full factorial central composite rotary design for these four independent variables (at five levels each) was employed to fit a polynomial model; which indicated that 30 experiments were required for this procedure. An optimization of CDKA; 1 and CycD6; 1 production levels in the soluble fraction was achieved. Protein conformation and stability were studied by circular dichroism and fluorescence spectroscopy. Finally, in vitro Cyc-CDK complex formation and its kinase activity were confirmed.