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. 2020 Jun 24;5(3):145-154.
doi: 10.1016/j.synbio.2020.06.004. eCollection 2020 Sep.

Silk fibroin as an additive for cell-free protein synthesis

Marilyn S Lee  1 , Chia-Suei Hung  2 , Daniel A Phillips  3 , Chelsea C Buck  2   3 , Maneesh K Gupta  2 , Matthew W Lux  1
Affiliations

Silk fibroin as an additive for cell-free protein synthesis

Marilyn S Lee et al. Synth Syst Biotechnol. .

Abstract

Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions. Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing, point-of-need manufacturing, and responsive materials. Meanwhile, silk fibroin from the silk worm, Bombyx mori, has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes. In this work, we explore the effects of silk fibroin as an additive in cell-free protein synthesis (CFPS) reactions. Impacts of silk fibroin on CFPS activity and stability after drying, as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed. We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%, which we attribute to macromolecular crowding effects. However, we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes. Further, the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture. Crosslinking attempts did not impact CFPS activity, but did yield localized protein aggregates rather than a hydrogel. We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.

Keywords: CFPS; CFPS, Cell-free Protein Synthesis; Cell-free protein synthesis; Cell-free systems; HRP, horse radish peroxidase; Preservation; SEM, Scanning Electron Microscopy; SF, Silk Fibroin; Silk fibroin; sfGFP, superfolder green fluorescent protein.

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Conflict of interest statement

The authors have no competing interests to declare.

Figures

Fig. 1
Fig. 1
Flow chart representation of SF processing to achieve different materials. SF from silk worm cocoons is isolated and solubilized. Enzymatic crosslinking at tyrosine residues to forms a hydrogel. Either SF solution or hydrogel forms a porous film or sponge when dried. CFPS reaction components are entrapped in SF by mixing with the concentrated SF solution before crosslinking or drying.
Fig. 2
Fig. 2
sfGFP production by CFPS in the presence of SF. (a) Comparison of relative sfGFP fluorescence at the endpoint of a CFPS reaction at 8 h. (b) Comparison of time to half maximal sfGFP fluorescence. Black bars are reactions where only SF is added. Grey bars are reactions containing SF as well as crosslinking reagents HRP and H2O2. Error bars represent the 95% confidence interval, n ≥ 4. (* indicates p < 0.05 compared to 0% (w/v) SF).
Fig. 3
Fig. 3
Freezing and lyophilization of CFPS reactions with SF solution (solution) or hydrogel (crosslinked). (a) Effects of freezing and lyophilization on CFPS activity measured by end point sfGFP fluorescence with and without presence of SF or HRP and H2O2. Error bars represent the 95% confidence interval, n ≥ 4. (* indicates p < 0.05 compared to 0% (w/v) SF) (b) Image of lyophilized SF fibroin crosslinked with HRP and H2O2 at various SF concentrations (c) Image of the samples in (b) after rehydration, total volume 100 μL.
Fig. 4
Fig. 4
Stability of lyophilized SF-CFPS reactions to time and temperature. (a–c) Mean endpoint sfGFP levels of CFPS reactions containing 0% (grey) or 1% (w/v) (black) SF lyophilized, exposed to one of three temperatures (a = room temperature, b = 37 °C, c = 50 °C), and rehydrated. n ≥ 2. d) Comparison of drying configurations for air-dried CFPS reactions. Reagent mix and lysate were either dried together or separately. e) Effects of air-drying and extended storage times on CFPS activity in the presence of SF solution or SF with HRP and H2O2. For each reaction the lysate and reagent mix were dried separately. The first row of labels shows whether water (W) or SF (S) was added before drying with a percentage to indicate the final concentration of SF. The supplemented component (Supp. compt.) refers to the component to which SF or water was added, either the lysate or the reagent mix. Error bars represent the 95% confidence interval, n ≥ 3.
Fig. 5
Fig. 5
Microplate reader assay to detect appearance of dityrosine fluorescence. (a) Samples with or without CFPS, 1% (w/v) SF, or HRP and H2O2. Error bars represent the 95% confidence interval. (b) Samples with SF, with or without CFPS, and with increasing levels of crosslinking reagents HRP and H2O2.
Fig. 6
Fig. 6
SF-CFPS morphology imaged via fluorescent microscopy and SEM. (a–f) sfGFP fluorescence of hydrated samples (scale bar 20 μm): (a) CFPS reaction containing 1% (w/v) SF, HRP, and H2O2; (b) CFPS reaction containing HRP and H2O2; (c) CFPS reaction; (d) purified sfGFP mixed with 1% SF, HRP, and H2O2; (e) background fluorescence with 1% SF, HRP, and H2O2; (f) CFPS reaction, 1% (w/v) SF, no crosslinking reagents. Images in panels (a), (c), (e), and (f) were collected at 800–850 V detector gain and 2.0 digital gain while panel (d) was collected at 650 V detector gain and 1.0 digital gain. Panel (b) has an asterisk because the image was collected using a different microscope as described in methods. (g–j) SEM images of lyophilized samples: (g, h) 1% (w/v) SF, HRP, H2O2 (f = scale bar 200 μm, g = scale bar 50 μm); (i, j) CFPS reaction, 1% (w/v) SF, HRP, H2O2 (h = scale bar 200 μm, i = scale bar 50 μm).
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