Cost-effective large-scale expression of proteins for NMR studies

Abstract

We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling ofH, C andN using auto-induction or isopropyl-β-D-1-thiogalactopyranoside-induction are described, with primary focus on minimal isotope consumption and high reproducibility of protein expression. These protocols are based on high cell-density fermentation, but the key procedures are easily transferred to shake flask cultures.

Pulsed Electron-Electron Double Resonance (PELDOR) and Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy in Bioanalysis

“…NMR studies of proteins usually require the incorporation of NMR-active isotopes as nitrogen 15 N and carbon 13 C, and sometimes deuterium 2 H, to improve the spectral quality and facilitate the interpretation of the spectra. To produce isotopic labeled samples, a variety of expressing systems can be used, from bacterial, yeast or insect cells, to cell-free expression (Dutta et al 2012;Franke et al 2018;Klopp et al 2018;Rosenblum and Cooperman 2014). The size and complexity of the target protein determines the labeling scheme to be used (Ohki and Kainosho 2008;Zhang and van Ingen 2016).…”

Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins

“…induction and/or harvesting) to be matched. On the other hand, if cell concentration is increased to reach a higher yield by using a fermenter for example, then normal size laboratory equipment as well as improved and tunable biotechnology protocols can be applied (Klopp et al, 2018). However, the close monitoring of the pH, air flux and the concentration of selected metabolites requires a disciplined and automation-oriented technology.…”

“…Cai et al (1998) used a fed-batch fermentation process for 15 N-and 13 C/ 15 N-labelling, in which the cells were grown in the presence of unlabelled isotopes in the initial phase. In a comprehensive study, several fermentation protocols were implemented with auto-or IPTG-inducing on minimal or rich media (Klopp et al, 2018).…”

Increasing the buffering capacity of minimal media leads to higher protein yield

“…Early examples of acclimatizing bacteria include growing cells on agar plates or in liquid culture with increasing ratios of D 2 O:H 2 O (Venters et al 1995;Gardner and Kay 1998). Recently, several reports have appeared describing new methods for increasing protein yield using higher cell densities, fermentation techniques, or amino acid drop-out algae lysate supplements (Cai et al 2016;O'Brien et al 2018;Klopp et al 2018). Whereas impressive gains in protein expression are noted in each, many of these methods require special equipment (e.g., fermenters) and none are as simple as the typical minimal media and methods for heterologous protein expression.…”