Spatio‐Temporal Photoactivation of Cytotoxic Proteins

Abstract Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non‐cell surface targets due to their poor cellular uptake. While cell‐penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio‐temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.

for 15 minutes. This cycle was repeated three times. The dried solid was dissolved in dry dichloromethane (150 mL) and the solution cooled to 0 ºC for 30 minutes. Phosphorous tribromide (1.16 mL, 12.3 mmol, 0.4 eq.) was added dropwise over 10 minutes maintaining the temperature of the solution at 0 ºC. Pyridine (0.2 mL, 2.5 mmol, 0.08 eq.) was then added to the reaction mixture. The solution was stirred at 0 ºC for 15 minutes and then at room temperature for 1.5 hours. The reaction was then cooled to 0 ºC again and methanol (7 mL) was added. The solution was stirred at room temperature for 30 minutes. The solvent was then removed under reduced pressure and the residue was partitioned between dichloromethane and saturated sodium bicarbonate solution. The aqueous phase was extracted with further dichloromethane (2 x 50 mL) and the combined organic layers were dried over magnesium sulfate, filtered and the solvent was removed under reduced pressure. The product was purified by column chromatography (1:1 hexane:dichloromethane) to yield 5 as a yellow solid (5.62 g, 67% yield). The product displayed spectroscopic data consistent with those reported previously.  (1). A solution of (R,S)-1-bromo-1-[4',5'-(methylendioxy)-2'nitrophenyl]ethane (5, 5.0 g, 18.2 mmol, 1.1 eq.) in degassed dioxane (20 mL) was added dropwise to a degassed solution of Lcysteine (2.0 g, 16.5 mmol, 1.0 eq.) in sodium hydroxide (33 mL, 0.5 M) over 1 hour. The reaction mixture was stirred at room temperature overnight. Salts formed during the reaction were filtered off and the solution was acidified with hydrobromic acid until precipitation of the product was observed. The product was filtered off to yield 1 as a light yellow solid (2.2 g, 40% yield). The product displayed spectroscopic data consistent with those previously reported.

Plasmid construction
Amino acid sequences for intein-split mCherry, PCC2RS synthetase and intein-split saporin-S6 are detailed in Table S1. The pET30b(+) expression plasmid for (144TAG)-mCh-intein-erry with C-terminal His 6 affinity tag, codon-optimised for expression in Escherichia coli, was purchased from AddGene. A TAG144C mutant was generated by site-directed mutagenesis using the primers defined in Table S2. Cell-penetrating peptide (CPP) sequences were cloned into both plasmids by PCR using the primers described in Table S2. CPP sequences were ligated to the plasmid by Gibson assembly for -HA2-TAT or Golden Gate assembly for -TAT, -cTAT and -HA2-cTAT.
Trans-splicing constructs were generated by PCR amplification from the SDM generated plasmid containing (144Cys)-mCh-inteinerry using the primers described in Table S2, which attached complementary BsaI sites to expression vectors pETM11 (C-terminal construct) and pET19 (N-terminal) for Golden Gate assembly. Both vectors encode for a N-terminal His x6 affinity tag, and pETM11 contained a TEV (Tobacco Etch Virus) cleavage site after the affinity tag.
The pUC plasmid of the orthogonal tRNA synthetase, PCC2RS, was kindly provided by Dr. Yu-Hsuan Tsai (Cardiff University, UK). Three copies of PCC2RS were amplified by PCR using the primers found in Table S2, which attached four different complementary BsaI restriction sites for Golden Gate assembly to pCDF expression vector pCDF.
DNA sequences encoding saporin-S6 and barnase, flanked by BsaI sites in 3' and 5' ends, codon-optimised for expression in E. coli, were purchased from Thermofisher GeneArt TM as shown in Table S1. Products were independently ligated to the pET30b(+) expression plasmid with the -HA2-cTAT CPP sequence by Golden Gate assembly.
PCR amplifications were performed variously with Pfu polymerase (New England BioLabs UK Ltd) or PrimeSTAR (Takara Bio Europe). PCR products were purified by agarose gel and ligated by Gibson assembly (Gibson assembly mix from New England BioLabs UK Ltd) or Golden Gate assembly (Using BsaI and T4 ligase from New England BioLabs UK Ltd). All gene sequences were confirmed by sequencing from the T7 promoter and/or the T7 termination sequence (Eurofins Scientific, UK).

Protein expression and purification
mCherry-CPP constructs. Proteins were expressed using BL21 (DE3) E. coli (Merck) cells transformed with the plasmid coding for the appropriate (144Cys)-mCherry-CPP constructs grown in LB media supplemented with kanamycin (50 µg/ml). Cultures were grown at 37 ºC until optical density at 600 nm (OD 600 ) equaled or exceeded 0.6, then expression was induced with isopentenyl thiogalactose (0.5 mM, IPTG, Melford) and the cultures were incubated at 25 ºC overnight. Cells were harvested by centrifugation and cell pellets were stored at -20 ºC until required. mCherry, mCherry-TAT and mCherry-cTAT were purified by suspending the cell pellets in lysis buffer (20 mM Tris, 30 mM NaCl, 20 mM imidazole, pH 8.0) supplemented with egg hen white lysozyme (1 mg/mL, Sigma Aldrich) and phenylmethylsulfonyl fluoride (1 mM, PMSF, Melford). Cells were lysed by sonication (5 s on, 10 s off for a total of 5 min active time) and centrifuged (38,000 x g for 45 min at 4 ºC) to remove cell debris. The supernatant was loaded onto a Ni-NTA column and a step-wise gradient of imidazole was applied (20, 30, 60, 100, 300, 500 mM). Proteins were eluted mostly pure in 60-300 mM imidazole fractions. Imidazole was removed by dialysis against lysis buffer. TEV-His 6 protease and dithiothreitol (DTT) to a final concentration of 5 mM were added to the CPP constructs to remove the C-terminal His 6 tag (overnight digestion at 4 ºC). The solution was passed through a Ni-NTA column to remove the protease and the desired protein was collected in the flow-through. Samples were concentrated to 10 µM (concentration determined by absorbance, ε = 72,000 M -1 ·cm -1 ) by centrifugal filtration and buffer-exchanged in DMEM (Dulbecco's Modified Eagle Medium, Fisher Scientific) media for the cell assays.
mCherry-HA2-TAT and mCherry-HA2-cTAT were purified by suspending the cell pellets in denaturing lysis buffer (20 mM Tris, 8 M urea, 30 mM NaCl, 20 mM imidazole, pH 8.0) supplemented with PMSF (1 mM). Cells were lysed by sonication (5 s on, 10 s off for a total active time of 5 min) and centrifuged (38,000 x g for 45 min at 4 ºC) to remove cell debris. Supernatant was loaded onto a Ni-NTA column and a step-wise gradient of imidazole was applied (20, 30, 60, 100, 300, 500 mM). Protein was eluted mostly pure in 60-300 mM imidazole fractions. Proteins were refolded by removing urea and imidazole by dialysis against lysis buffer. TEV-His 6 protease and DTT (dithiothreitol) were added to a final concentration of 5 mM to remove the C-terminal His x6 tag (overnight digestion at 4 ºC). The solution was loaded onto a Ni-NTA column and the flow-through containing the protein was collected. Samples were concentrated to 10 µM (concentration determined by absorbance, ε = 72,000 M -1 ·cm -1 ) by centrifugal filtration and buffer-exchanged in DMEM (Dulbecco's Modified Eagle Medium, Fisher Scientific) media for the cell assays. Protein constructs were filter-sterilised with 0.22 µm Millex syringe filter into sterile microcentrifuge tubes, and tubes were stored at -80 ºC until required.
Trans-splicing constructs. Cells were transformed with the appropriate plasmid for the trans-splicing constructs and grown in LB media supplemented with ampicillin (100 µg/ml) for the N-terminal construct or kanamycin (50 µg/ml) for the C-terminal construct. Cultures were grown at 37 ºC until OD 600 = 0.6, then expression was induced with IPTG (0.5 mM) and the cultures incubated at 25 ºC overnight. Cells were harvested by centrifugation and cell pellets were stored at -20 ºC until required.
Both constructs were purified by suspending the cell pellets in denaturing lysis buffer supplemented with PMSF (1 mM). Cells were lysed by sonication (5 s on, 10 s off for 5 min total active time) and centrifuged (38,000 x g for 45 min at 4 ºC). The supernatant was loaded in Ni-NTA column and a step-wise gradient of imidazole was applied (20, 30, 60, 100, 300, 500 mM). Protein was eluted mostly pure in 60-300 mM imidazole fractions. Constructs were refolded, and imidazole was removed, by step-wise dialysis against lysis buffer with decreasing concentrations of urea (4 M urea, 2 M urea, no urea). TEV-His 6 protease and DTT (dithiothreitol) to a final concentration of 5 mM was added to C-terminal construct remove the N-terminal His 6 tag and incubated overnight at 4 ºC. Samples were loaded to a Ni-NTA column and the flow-through containing the protein was collected. Samples were concentrated to 50 µM (concentration determined by Bradford assay) by centrifugation and buffer-exchanged into splicing buffer (20 mM Tris, 300 mM NaCl, 2 mM EDTA, pH 7.3) for kinetic assays.

Splicing kinetics assays
Irradiation studies. (ONB)-mCh-intein-erry (5 µM, in splicing buffer) was irradiated in a quartz cuvette (1 mm path length) by 365 nm light from a UVP Benchtop 2UV transilluminator (~4 mW/cm 2 ) for specified times. The contents of the cuvette were transferred to black 24-well plates for measuring fluorescence.
Fluorescence spectroscopy by plate reader. Samples were transferred to black 24-well plates and mCherry fluorescence was measured using a VICTOR X5 Multilabel Plate Reader (PerkinElmer) using a 579/25 excitation filter and a 615/8.5 emission filter. Data analysis was performed in Microsoft Excel. Concentrations of mature mCherry were calculated from a calibration curve performed at know concentrations of mCherry determined by absorbance. Main Text Paragraph.   Figure S3. Incubation studies of mCherry-cTAT. (A) 250,000 cells per well of a 24 clear well-plate were incubated for 1 to 8 hours with mCherry-cTAT (10 µM) in DMEM (serum free). Cells were washed three times with heparin in PBS and once with PBS before detaching them with trypsin and resuspending them in PBS (450 µL) and analyzing by FACS. (B) 250,000 cells per in a 24 clear well-plate) were incubated for 2 hours with mCherry-cTAT in varying concentrations from 5 to 30 µM in DMEM (serum free). Cells were washed three times with heparin in PBS and once with PBS before detaching them with trypsin and resuspending them in PBS (450 µL) and analysing by FACS. (C) 20,000 cells per well in a 96 black well-plate were incubated for 1 to 8 hours with mCherry-cTAT (10 µM) in DMEM (serum free). Cells were washed three times with heparin in PBS and once with PBS before adding DMEM supplemented with 10% FBS (100 µL) and Celltiter Blue reagent (20 µL). Cells were incubated with the reagent for 4 hours and fluorescence at 610 nm was measured using a plate reader. Untreated cells were kept in DMEM (supplemented with 10% FBS) during all the experiment, and no heparin washes were performed on them. All values were normalized to non-treated cells (considered to be 100% viability). Triton X-100 (0.01%) was employed as positive control for cell death. (D) 20,000 cells per well in a 96 black well-plate) were incubated for 2 hours with mCherry-cTAT in concentrations varying from 5 to 30 µM in DMEM (serum free). Cells were washed three times with heparin in PBS and once with PBS before adding DMEM supplemented with 10% FBS (100 µL) and Celltiter Blue reagent (20 µL). Cells were incubated with the reagent for 4 hours and fluorescence at 610 nm was measured by a plate reader. Untreated cells were kept in DMEM (supplemented with 10% FBS) during all the experiment, and no heparin washes were performed on them. All values were normalized to non-treated cells (considered to be 100% viability). Triton X-100 (0.01%) was employed as positive control for cell death.         Figure S3. Incubation studies of mCherry-cTAT. (A) 250,000 cells per well of a 24 clear well-plate were incubated for 1 to 8 hours with mCherry-cTAT (10 µM) in DMEM (serum free). Cells were washed three times with heparin in PBS and once with PBS before detaching them with trypsin and resuspending them in PBS (450 µL) and analyzing by FACS. (B) 250,000 cells per in a 24 clear well-plate) were incubated for 2 hours with mCherry-cTAT in varying concentrations from 5 to 30 µM in DMEM (serum free). Cells were washed three times with heparin in PBS and once with PBS before detaching them with trypsin and resuspending them in PBS (450 µL) and analysing by FACS. (C) 20,000 cells per well in a 96 black well-plate were incubated for 1 to 8 hours with mCherry-cTAT (10 µM) in DMEM (serum free). Cells were washed three times with heparin in PBS and once with PBS before adding DMEM supplemented with 10% FBS (100 µL) and Celltiter Blue reagent (20 µL). Cells were incubated with the reagent for 4 hours and fluorescence at 610 nm was measured using a plate reader. Untreated cells were kept in DMEM (supplemented with 10% FBS) during all the experiment, and no heparin washes were performed on them. (D) 20,000 cells per well in a 96 black well-plate) were incubated for 2 hours with mCherry-cTAT in concentrations varying from 5 to 30 µM in DMEM (serum free). Cells were washed three times with heparin in PBS and once with PBS before adding DMEM supplemented with 10% FBS (100 µL) and Celltiter Blue reagent (20 µL). Cells were incubated with the reagent for 4 hours and fluorescence at 610 nm was measured by a plate reader. Untreated cells were kept in DMEM (supplemented with 10% FBS) during all the experiment, and no heparin washes were performed on them.            CATTGGTCTCTTATGGCACAGGTGATTAATACCTTTGATGGTGTTGCCGATTATCTGCAGA  CCTATCATAAACTGCCGGATAACTATATCACCAAAAGCGAAGCACAGGCATTAGGTTGGG  TTGCATAGCTGAGCTATGAAACCGAAATTCTGACCGTTGAATATGGTCTGCTGCCGATTG  GTAAAATTGTGGAAAAACGTATTGAGTGCACCGTGTATAGCGTGGATAATAACGGTAACA  TTTATACCCAGCCGGTTGCACAGTGGCATGATCGTGGTGAACAAGAAGTTTTTGAATACT  GTCTGGAAGATGGTAGCCTGATTCGTGCAACCAAAGATCACAAATTTATGACCGTGGATG  GTCAGATGCTGCCCATTGATGAAATTTTTGAACGTGAACTGGATCTGATGCGTGTTGATAA  TCTGCCGAATATCAAAATTGCGACCCGTAAATATCTGGGCAAACAGAACGTGTATGATATT  GGTGTGGAACGCGATCATAATTTCGCACTGAAAAATGGTTTTATCGCCAGCAATTGCAAA  GGTAATCTGGCAGATGTTGCACCGGGTAAAAGCATTGGTGGTGATATCTTTAGCAATCGC  GAAGGTAAACTGCCTGGTAAAAGCGGTCGTACCTGGCGTGAAGCAGATATCAATTATACC  AGCGGTTTTCGTAATAGCGATCGCATTCTGTATAGCAGCGATTGGCTGATCTATAAAACC  ACCGATCATTATCAGACCTTCACCAAAATTCGCGAGAAGAGACCTTAC  Table S2. Primers used to generate plasmids.