A Light‐Activatable Photocaged Variant of the Ultra‐High Affinity ALFA‐Tag Nanobody

Abstract Nanobodies against short linear peptide‐epitopes are widely used to detect and bind proteins of interest (POI) in fusion constructs. Engineered nanobodies that can be controlled by light have found very recent attention for various extra‐ and intracellular applications. We here report the design of a photocaged variant of the ultra‐high affinity ALFA‐tag nanobody, also termed ALFA‐tag photobody. ortho‐Nitrobenzyl tyrosine was incorporated into the paratope region of the nanobody by genetic code expansion technology and resulted in a ≥9,200 to 100,000‐fold impairment of the binding affinity. Irradiation with light (365 nm) leads to decaging and reconstitutes the native nanobody. We show the light‐dependent binding of the ALFA‐tag photobody to HeLa cells presenting the ALFA‐tag. The generation of the first photobody directed against a short peptide epitope underlines the generality of our photobody design concept. We envision that this photobody will be useful for the spatiotemporal control of proteins in many applications using cultured cells.

. Flow cytometry analysis of E. coli cells presenting ALFA-Pb without (-UV) and with (+UV) irradiation with light (λ=365nm) and incubation with sfGFP-ALFA-tag. Controls show uninduced cell population that have not expressed the ALFA-Pb (gray signal covered behind red signal). UT5600 cells [1b, 2] were used and were co-transformed with the respective expression plasmid and the pEVOL-ONBY plasmid. Cells were cultured at 37 °C in LB-medium (600 mL) with the corresponding antibiotic(s) (100 µg/mL ampicillin, 34 µg/mL chloramphenicol) until an OD600 of 0.7 -0.9 was reached. The temperature was then shifted to 28 °C and protein expression was induced for 4 h by adding IPTG (0.4 mM final concentration). For suppression conditions, 0.5 mM o-(2-nitrobenzyl)-L-tyrosine (ONBY), presolved in 1 M NaOH, and 0.2% arabinose were added, and cells were incubated for 4 h at 37 °C. Cells were pelleted by centrifugation and resuspended in Ni-NTA buffer (50 mM Tris/HCl, 300 mM NaCl, pH 8.0). Resuspended cells were ruptured using sonification. Insoluble material was removed by centrifugation and the supernatant fractions were used to purify the proteins. Purification of His-tagged proteins via Ni-NTA affinity chromatography was performed at 4 °C using gravity flow columns with a bed volume of 1.0 mL of Ni-NTA resin (Cube Biotech) pre-equilibrated with Ni-NTA buffer with 20 mM imidazole. Following loading of the supernatant fractions (with 20 mM imidazole), three steps of washing with five to ten column volumes (cv) of Ni-NTA buffer (with 20 mM imidazole) were performed. Proteins were eluted with 4 mL Ni-NTA buffer (with 250 mM imidazole) fractions containing the desired protein were pooled and concentrated in Vivaspin® Turbo 4 concentrator spin columns (Sartorius, Göttingen, Germany) to ~¼ of the volume.

Recombinant gene expression and protein purification
As a second purification step a size exclusion chromatography was performed on an ÄKTA Purifier System (GE Healthcare) with a Superdex200 column and a flow rate of 1 mL/min in PBS buffer (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2PO4, 8.1 mM Na2HPO4, pH 7.4). Protein elution was monitored by absorption at 280 nm and collected fractions were analyzed by SDS-PAGE. Purified fractions were combined, 10% glycerol (v/v) was added, concentrated in Vivaspin® Turbo 4 concentrator spin columns (Sartorius, Göttingen, Germany), flash frozen in liquid nitrogen and stored at -80 °C. Protein concentrations were determined using the calculated extinction coefficient at 280 We achieved a yield of 0.58 mg purified protein per liter of E. coli cell culture medium for the ALFA-Nb expressed in the periplasm. For suppression of ALFA-Pb we achieved 0.32 mg/L (55 % efficiency).

Protein bioconjugation with AlexaFluor647
Nanobody and photobody proteins were chemically modified using AlexaFluor647 maleimide (Invitrogen, Carlsbad, USA). A final concentration of 20 µM protein in PBS buffer was incubated with 50 µM TCEP for 15 min at RT before labeling to reduce potentially oxidized cysteines. AlexaFluor647 maleimide was then added in 5x excess and incubated for 30 min in the dark at RT. This step was repeated once. To quench the reaction 2 mM DTT was added and incubated for 10 min in the dark at RT. The labeled protein was purified by Ni-NTA affinity chromatography and concentrated using Vivaspin® Turbo 4 concentrator spin columns to respective concentrations.

Fluorescence and Coomassie-stained SDS-PAGE gel images
Fluorescence SDS-PAGE gel images were captured on a Typhoon FLA 9500 laser scanner (GE Healthcare) before Coomassie-staining. Fluorescence stage was used with a 635 nm laser and "Alexa Fluor 647"-method for detection. Coomassie-stained SDS-PAGE gel images were captured on a CanonScan 9000F Mark II system.

UV irradiation of cells using an LED lamp
For UV irradiation of cells before fixing with PFA an LED lamp was used (M365LP1-365 nm; Thorlabs Inc, Newton, USA). An LED power of 530 mW was determined for the instrument using a thermophile sensor (A3, P/N 7Z02621, Aperture: Ø 9.5 mm) (Ophir Optronics Solutions Ltd., Jerusalem, Israel).
Samples were irradiated for the indicated time periods with an irradiation distance of 5 cm.

Nanobody display on cell surface
Nanobody encoding genes were cloned into a pBAD-vector for the AIDA autodisplay system [1,3] using E. coli DH5α cells as cloning host. E. coli BL21 Gold (DE3) cells were co-transformed with the respective AIDA plasmid (Table S2) and, if applicable, with the pEVOL-ONBY plasmid for incorporation of o-(2-nitrobenzyl)-L-tyrosine (ONBY). Presenting E. coli cells were prepared by inoculating LB medium (20 mL) with respective antibiotics with an overnight culture in the ratio 1:20.
Bacterial cell cultures were then grown at 37 °C with shaking (180 rpm) until an OD600 of 0.5 was reached. In case of suppression conditions, ONBY (3 mM final concentration) was added to the medium and gene expression was induced with 0.4% arabinose. Induced cells were cultured for 3 h at 30 °C in case of regular expression without amber stop codon suppression or for 2 h at 37 °C in case of suppression with the unnatural amino acid. The cells were then washed three times with ice cold PBS buffer (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2PO4, 8.1 mM Na2HPO4, pH 7.4; sterile-filtered) using a centrifugation step (5000 g) at 4 °C for 2 min to pellet the cells, and then stored after resuspension in

Determination of binding affinity of nanobodies presented on cell surface by flow cytometry
Nanobody presenting E. coli cells were washed with ice cold PBS buffer (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4; sterile-filtered), pelleted by centrifugation (5000 g for 2 min at 4 °C) and were then resuspended in PBS buffer and diluted to OD600 = 1.0. For photodeprotection, 100 µL cells (OD600 = 1) were transferred into a thin-walled PCR tube (Greiner Bio-One,

Microscale thermophoresis (MST)
A dilution series with 16 aliquots of the ALFA-photobody (1) was prepared in PBS buffer before measurement. 10 µL of fluorescent sfGFP-ALFA-tag protein (10 nM final concentration) was mixed with 10 µL of the 16 photobody aliquots and incubated for 15 min at 25 °C. MST measurements were performed on a Nanotemper Monolith NT.115 with standard coated capillaries (NanoTemper Technologies, Munich, Germany). The blue LED power was at 100% and the MST power was at 80%.
Measured data points were exported and plotted against the log (concentration) of nanobody to determine KD by nonlinear fitting.

Cell culture techniques and cell surface labeling
HeLa cells were cultured in EMEM (supplemented with 10% fetal calf serum, 1% non-essential amino acids and 1% L-glutamine) at 37 °C and 5% CO2. 50% confluent cells were used for transient transfection with the plasmid encoding HA-ALFA-tag-Trx-TMD-mCherry using calcium phosphate precipitation technique on a 24 mm coverslip in a 35 mm cell S7 culture dish, followed by 16 to 20 h of incubation. Table S3 lists the construct used for transient transfections.
For labeling of the cell surface, cells were washed two times with PBS and 1 mL of a 10 nM solution of the AlexaFluor647 modified nanobody or photobody in EMEM was added. Incubation was performed at room temperature for 15 minutes.

Confocal laser scanning microscopy
Cells were fixed with 4% paraformaldehyde in PBS for 20 min at 25 °C, washed three times with PBS, stained with DAPI and mounted on coverslips using Aqua/Poly-Mount mounting solution (Polysciences). Confocal microscopy was carried out using a 63X water-immersion objective lens on a Leica DMi8 system. DAPI laser (405 nm), mCherry laser (552 nm), Alexa647 laser (638 nm).

Mass spectrometry
Mass analyses of intact proteins were performed using an UltiMate™ 3000 RS system (Thermo Fisher Scientific GmbH, Dreieich, Germany) connected to a maXis II UHR-qTOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) with a standard ESI source (Apollo, Bruker Daltonik GmbH, Bremen, Germany). Proteins were reduced with 2 mM TCEP at room temperature for 10 minutes to avoid inhomogeneity issues. Then, samples were acidified using a 5% formic acid solution to reach a pH 2-3 and centrifuged (14000 rpm, 3 min). According to the protein concentration, an appropriate volume of the supernatant was loaded on a C4 column (Advance Bio RP-mAb C4, 2.1 mm x 50 mm, 3.5 µm, Agilent Technologies, Waldbronn, Germany) at a flow rate of 0.6 mL/min in 5% eluent B (eluent A: 0.1% formic acid in water; eluent B: 0.1% formic acid in acetonitrile). After a desalting period of 7 minutes at 5% B, a steep gradient was applied (5-60% B in 2 min). MS settings: capillary voltage 4500 V, end-plate offset 500 V, nebulizer 5.0 bar, dry gas 9.0 L/min, dry T=200°C, mass range m/z 300-3000. Data were analyzed with DataAnalysis 4.4 (Bruker Daltonik GmbH, Bremen, Germany) and deconvolution was performed using the MaxEnt algorithm implemented in the software. Unless otherwise mentioned, the averagine-based SNAP algorithm was employed to identify peaks and to calculate the monoisotopic masses.