E. coli "Stablelabel" S30 lysate for optimized cell-free NMR sample preparation

Abstract

Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of ¹⁵N labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative "Stablelabel" containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in "Stablelabel" lysates. By taking advantage of ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.

Keywords: Cell-free expression; Metabolic engineering; NMR of membrane proteins; Protein labeling; S30 lysate; Stable isotope scrambling.

Fig. 1

Action of inhibitors used to suppress amino acid conversions in E. coli S30 CF lysates

Fig. 2

Analysis of amino acid scrambling via GFP expression. GFP expression in standard A19 S30 CF lysates carried out either in presence of all amino acids (all) or in absence of the indicated amino acids. Data normalized to GFP concentration of 3.5 mg/mL Quantification was carried out via fluorescence measurement in the RM after harvesting of the CF reaction. Bars represent means of expression yields obtained from at least 3 independent CF reactions

Fig. 3

Growth kinetics and CF protein production performance of S30 lysates from selected A19 mutants. A Growth curves of E. coli A19 WT and mutants cultivated in a 15 L stirred-tank bioreactor in 10 L YPTG medium at 37 °C, 500 rpm. 1 g/L L-Gln was supplied to the culture medium to restore growth of glnA mutants (+ Q). Standard YPTG culture medium contained 0.1 M glucose (+ G), except for some glmS mutants such as M 1-3-4-5-6-7 (−G) where no glucose was added due to growth problems. Furthermore, glmS mutant cultures were supplied with additional 1 g/L N-acetyl D glucosamine (+ GlcNAc). B Extract performance was evaluated by expression of GFP under standard conditions. Quantification was carried out via GFP fluorescence measurement in the CF reaction mixture. Bars represent means of expression yields obtained from at least three independent CF reactions

Fig. 4

Selective CF labeling of CypD (43–207) with either A19 or “Stablelabel” S30 lysates. Spectra overlays illustrate results of CypD selective labeling with ¹⁵N L-Asp (a), ¹⁵N L-Glu (b), ¹⁵N L-Asn (c) or ¹⁵N L-Gln (d) using either A19 (red) or “Stablelabel” (blue) S30 lysates. The yellow spectrum in b was obtained after addition of 20 mM AOA in the CF reaction to suppress L-Glu to L-Ala label conversion. The yellow spectrum in d was obtained after incubation of CF lysate with 5 mM DON at RT for 1 h before CypD expression. [¹⁵N, ¹H] BEST-TROSY spectra were measured on CypD samples of 100–150 µM concentration in sodium phosphate buffer pH 7.0 at 303 K. All A19 spectra were recorded on 900 or 950 MHz spectrometers while “Stablelabel” spectra were recorded on 600 or 700 MHz spectrometers

Fig. 5

¹⁵N L-Gln labeling of CypD CF expressed with lysates of either A19 (red), gltB M5 (yellow) or glmS M 1-3-4-6-7 (blue). Scrambling of L-Gln in mutant lysates confirms that several enzymes contribute glutaminase activity and that single mutants do not sufficiently reduce L-Gln to L-Glu conversion. [¹⁵N, ¹H] BEST-TROSY spectra were acquired with CypD samples of 100–150 µM in Na-phosphate, pH 7.0, at 303 K. A19 and M5 spectra were recorded on a 900 MHz, M 1-3-4-6-7 spectrum on a 700 MHz spectrometer, respectively

Fig. 6

Selective CF labeling of CypD (43–207) with “Stablelabel” S30 lysate with low contour levels. Blue spectra illustrate results of CypD selective labeling with ¹⁵N L-Asp (a), ¹⁵N L-Glu (b), ¹⁵N L-Asn (c) or ¹⁵N L-Gln (d) using “Stablelabel”. The yellow spectrum in b was obtained after addition of 20 mM AOA in the CF expression with “Stablelabel” to suppress L-Glu to L-Ala label conversion. The yellow spectrum in d was obtained after incubation of “Stablelabel” lysate with 5 mM DON at RT for 1 h before CypD expression. [¹⁵N, ¹H] BEST-TROSY spectra were measured on CypD samples of 100–150 µM concentration in sodium phosphate buffer pH 7.0 at 303 K. Spectra were recorded on 600 or 700 MHz spectrometers

Fig. 7

Amino acid synthesis in S30 lysates of either A19 or “Stablelabel”. GFP was CF expressed under standard conditions using the two different lysates either with all amino acids supplied or with L-Ala, L-Asp, L-Asn, L-Glu or L-Gln omitted from the reaction. Expression controls with inhibitors DM + AOA, AOA and DON were included. Control are values of reactions in presence of all 20 amino acids. All data are normalized to the A19 control value (1 = 3.5 mg/mL)

Fig. 8

Conversion of methyl labeled precursor KIV to L-Val and MOV to L-Leu. GFP expression was carried out either with A19 (white bars) or with “Stablelabel” (green bars) S30 lysates. a/c The first two bars indicate control expressions with both lysates with 1 mM L-Val/L-Leu in the reaction. The next two bars show GFP expression with L-Val/L-Leu replaced by 4 mM KIV/MOV. For MOV, the same reaction was carried out in presence of 20 mM AOA, an unselective transaminase inhibitor. The next bar (-Val/-Leu) indicates expression with A19 S30 lysate without L-Val/L-Leu. The next section of the graph shows GFP expression with L-Val/L-Leu replaced by converted KIV/L-Glu or MOV/L-Glu mixtures. The indicated molarities represent the theoretical L-Val/L-Leu concentration if all KIV/MOV was converted. b [¹³C, ¹H] XL-ALSOFAST-HMQC spectrum of PR in non-deuterated diC7PC labeled with L-Val (4-¹³C, 2, 3, 4, 4, 4,-D5) converted from KIV (3-methyl-¹³C, 3, 4, 4, 4-D4). Detergent signals are labeled with a “d” d [¹³C, ¹H] XL-ALSOFAST-HMQC spectrum of PR in non-deuterated diC7PC labeled with L-Leu (5-¹³C, 2, 3, 3, 4, 5, 5, 5-D7) converted from MOV (4-methyl-¹³C, 3, 3, 4, 5, 5, 5-D6). Spectra were acquired on a 950 MHz Bruker Avance Neo spectrometer at a temperature of 313 K. Conversion of both precursors was carried out in ~ 90% deuterated buffer to avoid proton incorporation at Cα during transamination by IlvE

Fig. 9

Conversion of KIV (3-methyl-¹³C, 3, 4, 4, 4-D4) to L-Val (3-methyl-¹³C, 2, 3, 4, 4, 4,-D5) and MOV (4-methyl-¹³C, 3, 3, 4, 5, 5, 5-D6) to L-Leu (4-methyl-¹³C, 2, 3, 3, 4, 5, 5, 5-D7) precursors by supplied IlvE. a Overlay of non-converted KIV (red), KIV after conversion with 75 mM KIV and 75 mM L-Glu (blue) and KIV after conversion with 75 mM KIV and 300 mM L-Glu (yellow). b Overlay of non-converted MOV (red) and MOV after conversion with 24 mM MOV and 100 mM L-Glu (blue). [¹³C, ¹H]-HSQC spectra were recorded at T = 313 K on a Bruker Avance II 500 MHz spectrometer, equipped with a room-temperature ¹H{¹³C/¹⁵N} three-axes gradient triple-resonance probe. Precursor conversion was performed in 100 mM Tris–HCl, pH 8.0, 100 mM NaCl dissolved in D₂O and 1 mg/mL IlvE. Upon completion of conversion, IlvE was removed by ultrafiltration and the mixtures were diluted 1:50 in 25 mM Na-acetate, pH 5.0 containing 5% D₂O and 0.15 mM DSS. Non converted precursors were measured under the same conditions