Title
Optimization of recombinant maize CDKA;1 and CycD6;1 production in Escherichia coli by response surface methodology
Author
Andrea A. E. Méndez, Liliana B. Pena, Lucrecia M. Curto, Marcelo D. Sciorra, Rita M. Ulloa, Sara M. Garza Aguilar, Jorge M. Vázquez Ramos, Susana M. Gallego
Year
2020
Journal
Protein Expression and Purification
Abstract
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.
Instrument
FP-6500, J-810
Keywords
Circular dichroism, Secondary structure, Tertiary structure, Fluorescence, Protein structure, Thermal stability, Protein folding, Biochemistry