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

Protein Expr Purif. 2020 Jan;165:105483. doi: 10.1016/j.pep.2019.105483. Epub 2019 Aug 31.

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.

Keywords: 1; Cell cycle proteins; Circular dichroism spectroscopy; Cyclin D6; Cyclin-dependent kinase; Zea mays L..

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CDC2 Protein Kinase / genetics*
  • CDC2 Protein Kinase / metabolism
  • Cyclins / genetics*
  • Cyclins / metabolism
  • Escherichia coli / genetics*
  • Gene Expression / drug effects
  • Isopropyl Thiogalactoside / metabolism
  • Models, Biological
  • Models, Statistical
  • Plant Proteins / genetics*
  • Plant Proteins / metabolism
  • Protein Conformation
  • Solubility
  • Temperature
  • Transfection
  • Zea mays / genetics*

Substances

  • Cyclins
  • Plant Proteins
  • Isopropyl Thiogalactoside
  • CDC2 Protein Kinase