Plant based production of myoglobin - a novel source of the muscle heme-protein

Abstract

Myoglobin is a heme-protein in the muscle of vertebrates with important functions in the oxygenation of tissues and as a regulator in nitric oxide signaling. Myoglobin from many species is also an important nutritional source of bioavailable iron. In this study, we have successfully produced human myoglobin in the leaves of Nicotiana benthamiana by transient expression using a viral vector delivered by Agrobacterium tumefaciens. Analyses confirmed that heme was incorporated and the protein was functional, with observed properties consistent with those of native myoglobins. A relatively high degree of purity could be achieved with low cost methods. The results show the high potential of plants as a production platform for heme proteins, a group of proteins of interest for iron nutrition applications and possible future pharmaceutical development.

Figure 1

Effects of Mb expression on the color (a) and absorbance (b) of N. benthamiana leaf extracts. (a) Image of protein extracts from agroinfiltrated leaves expressing myoglobin (left) and protein extracts from untreated leaves (right). (b) Absorbance spectra of clarified and filtered extracts: red: infiltrated, “cytosol accumulated” myoglobin construct, blue: infiltrated, “chloroplast targeted” myoglobin construct, green: Untreated N. benthamiana.

Figure 2

SDS-PAGE (a) and Western blot (b) analyses of N. benthamiana leaf extracts. Red arrows indicate putative Mb bands. Each sample was loaded first as the raw extract, then as heat treated extract following a short heat step (55 °C, 5 min, CO bubbled). Lanes 1–2: untreated sample; 3–4: agroinfiltrated, cytosol accumulated Mb; 5–6: agroinfiltrated, chloroplast targeted Mb; 7–8: agrosprayed, cytosol accumulated Mb; 9–10: agrosprayed, chloroplast targeted Mb. M: Seeblueplus2. Mm: MagicMark XP.

Figure 3

SDS-PAGE and Western blot analyses. (a) SDS-PAGE of protein extracts from N. benthamiana leaves showing the expressed Mb following agroinfiltration and its purity after each successive step of purification. Lane 1: control (extract of untreated leaves); Lane 2: raw extract; Lane 3: heated extract, Lane 4: concentrated (ca. 15x) and buffer exchanged extract, Lane 5: ammonium sulfate fractionated and buffer changed extract, Lane 6: anion exchange purified Mb. Note: 6 µl sample was loaded in lanes 1–3 and ~1 µl sample was loaded in lanes 4–6. (b,c) SDS-PAGE and Western blot of protein extracts from N. benthamiana leaves showing the expressed Mb following agrospray and its purity after each successive step of purification. Lane 1: control (extract of untreated leaves), Lane 2: raw extract, Lane 3: heated extract, Lane 4: concentrated extract (ca. 28x), Lane 5: ammonium sulfate fractionated and buffer changed extract, Lane 6: anion exchange purified Mb, Lane 7: anion exchange purified Mb (high conc. ~6 ng loaded). Note: 6 µl sample was loaded in lanes 1–3 and 2 µl in lanes 4–6. For whole figure: the total sample volume was kept roughly constant following the concentration step (lanes 4–6) to facilitate yield estimation. M: Seeblueplus2. Mm: MagicMark XP. (Full size gels are available in Supplementary Fig. S1).

Figure 4

UV-Vis absorbance spectra of Mb produced in the leaves of N. benthamiana, measured at 250–700 nm, with an enlarged section at 450–700 nm. oxy-Mb (red), carboxy-Mb (blue), deoxy-Mb (purple), met-Mb (green). Sodium dithionite absorbance dominates the spectrum below approximatively 390 nm for the displayed carboxy-Mb and deoxy-Mb spectra. (See Supplementary Table S1 for absorbance values for selected wavelengths).

Figure 5

Autoxidation test. (a) Autoxidation of Mb produced in N. benthamiana as followed by the decrease in normalized absorbance at 581 nm (average from three replicates, 95% confidence interval, showing the curve deviation). (b) Absorbance spectra (average of three samples) in 30 min intervals over 18 h. Arrows indicate overall trend over time.

Figure 6

Circular dichroism spectra of purified Mb protein produced in the leaves of N. benthamiana.

Figure 7

Temperature stability analysis (triplicates) by differential scanning fluorimetry. (a) oxy-Mb. (b) carboxy-Mb. The measured fluorescence ratio data and its corresponding first derivatives are displayed for each replicate. Both smoothed curves and data points are shown in the figure. Vertical dotted lines indicate the detected onset (gray) and melting temperatures (black) in the top graphs.

Figure 8

Change in absorption (409 nm) due to denaturation of plant produced Mb by Gu-HCl (95% conf. interval, n = 3).