Abstract
Fluorescent proteins often result in phototoxicity and cytotoxicity, in particular because some red fluorescent proteins produce and release reactive oxygen species (ROS). The photogeneration of ROS is considered as a detrimental side effect in cellular imaging or is proactively utilized for ablating cancerous tissue. As ancient textiles or biomaterials, silk produced by silkworms can directly be used as fabrics or be processed into materials and structures to host other functional nanomaterials. It is reported that transgenic fusion of far‐red fluorescent protein (mKate2) with silk provides a photosensitizer hybridization platform for photoinducible control of ROS. Taking advantage of green (visible) light activation, native and regenerated mKate2 silk can produce and release superoxide and singlet oxygen, in a comparable manner of visible light‐driven plasmonic photocatalysis. Thus, the genetic expression of mKate2 in silk offers immediately exploitable and scalable photocatalyst‐like biomaterials. It is further envisioned that mKate2 silk can potentially rule out hazardous concerns associated with foreign semiconductor photocatalytic nanomaterials.
Highlights
Fluorescent proteins often result in phototoxicity and cytotoxicity, in particular because some red fluorescent proteins (RFP) produce and release reactive oxygen species (ROS)
Cytotoxic RFP has been used as a means of selectively damaging specific proteins upon light activation, which is known as chromophoreassisted light inactivation (CALI).[6]
RFP is recapitulated as ‘genetically encoded ROS-generating proteins’ for inactivating target cells and ablating tissue of interest.[4d]. All of these characteristics of RFP suggest that semiconductor nanocrystals or conjugated nanoparticles for plasmonic photocatalysis can be replaced by phototoxic RFP
Summary
Materials For silkworm transgenesis for producing mKate silk, we used Bombyx mori bivoltine strain, Keumokjam (F1 hybrid between the Japanese parental line Jam 125 and the Chinese parental line Jam 140) from the National Institute of Agricultural Sciences (Wanju, Republic of Korea). The hatched larvae (i.e. silkworms) were reared in groups and fed with mulberry leaves under standard conditions (e.g. 25 ± 2 °C and 80 ± 10% relative humidity). We used the following chemicals as received: alcalase enzyme, dialysis tube (pore size 12,000 Da MWCO), dimethyl sulfoxide (DMSO; (CH3)2SO, 99%), dithiothreitol (DTT; C4H10O2S2, ≥ 98%), lithium bromide (LiBr, ≥ 99%), nitro blue tetrazolium chloride (NBT; C40H30Cl2N10O6, ≥ 98%), methylene blue (C16H18CIN3S, 0.05 wt.% in H2O), miracloth (pore size 22 – 25 m), phosphate buffered saline (PBS; pH 7.4), sodium azide (NaN3, ≥ 99.5%), sodium carbonate (Na2CO3, ≥ 99%), Triton X100, and 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA; C22H18O8, ≥ 90%) were purchased from SigmaAldrich Co. 4-[(9-acridinecarbonyl)amino]-2,2,6,6-tetramethylpiperidin1-oxyl (TEMPO-9-ac; C23H26N3O2, 95%) was purchased from Synchem UG & Co. KG (Altenburg, Germany). All experiments were performed under the ambient conditions (22 ± 2 °C and 40 ± 10% relative humidity)
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