Improving yields of deuterated, methyl labeled protein by growing in H2O

Abstract

Solution NMR continues to make strides in addressing protein systems of significant size and complexity. A fundamental requirement to fully exploit the N-H TROSY and C-H methyl TROSY effects is highly deuterated protein. Unfortunately, traditional overexpression in Escherichia coli (E. coli) during growth on media prepared in DO leads to many difficulties and limitations, such as cell toxicity, decreased yield, and the need to unfold or destabilize proteins for back exchange of amide protons. These issues are exacerbated for non-ideal systems such as membrane proteins. Expression of protein during growth in HO, with the addition of H-labeled amino acids derived from algal extract, can potentially avoid these issues. We demonstrate a novel fermentation methodology for high-density bacterial growth in HO M9 medium that allows for appropriate isotopic labeling and deuteration. Yields are significantly higher than those achieved in DO M9 for a variety of protein targets while still achieving 75-80% deuteration. Because the procedure does not require bulk DO or deuterated glucose, the cost per liter of growth medium is significantly decreased; taking into account improvements in yield, these savings can be quite dramatic. Triple-labeled protein is also efficiently produced including specific CH labeling of isoleucine, leucine, and valine using the traditional ILV precursors in combination with an ILV-depleted mix of H/N amino acids. These results are demonstrated for the membrane protein sensory rhodopsin II and the soluble proteins human aldoketoreductase AKR1c3, human ubiquitin, and bacterial flavodoxin. Limitations of the approach in the context of very large molecular weight proteins are illustrated using the bacterial Lac repressor transcription factor.

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

“…Therefore, our findings could be more general and subsequently extended to other bacterial cell growing protocols. For example, Wand and co-workers have described a method utilizing drop-out deuterated algal lysate (lacking isoleucine, leucine, and valine) for the production of highly deuterated, side chain methyl group labeled proteins in protonated medium (O'Brien et al 2018). While that described procedure utilized 1.5x buffered minimal media and produced substantial gains, our results suggest that even higher yields could be achieved with 2x buffered media.…”

“…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.…”

Characterization of Internal Protein Dynamics and Conformational Entropy by NMR Relaxation

“…Our laboratory has found that commercial protein refolding kits can greatly accelerate the identification of refolding conditions, even for large multi-domain proteins. More recently, we have developed a protocol for expression of proteins in E. coli during growth on H 2 O that results in extensive deuteration and does not require back-exchange (O’Brien, Lin, Fuglestad, Stetz, Gosse, Tommos et al, 2018). For quantitative backbone dynamics experiments, labeling schemes that place 13 C adjacent to amide 15 N should be avoided.…”

Alpha protons as NMR probes in deuterated proteins

“…d -amino acids may also inhibit bacterial growth at high concentrations (Hishinuma et al 1969 ; Bardaweel 2014 ). It was also noted that during growth on deuterated amino acid media (O’Brien et al 2018 ; Fiaux et al 2004 ), exchange of amide moieties occurs, but results in only 10–50% incorporation of alpha protons for hydrophobic residues (Löhr et al 2003 ).…”