A genetically encoded multifunctional unnatural amino acid for versatile protein manipulations in living cells

A multifunctional UAA, PABK, is developed for diverse protein manipulation purposes, especially protein activation in living cells.


General methods
All chemicals were obtained from J&K Co. unless otherwise specified. NMR spectra were recorded on a Bruker 400 MHz Fourier transform spectrometer at Peking University, China. 1 H and 13 C{ 1 H} NMR spectra were referenced to residual solvent. LC-MS analysis was performed using Waters ACQUITY UPLC I-Class SQD2 MS spectrometer with electrospray ionization (ESI). Compounds 1, 1 2 2 and trans-cyclooctenes 3 were prepared based on literature procedures. Monoclonal antibodies against myc-tag (#2276), p-Erk (#9101), Erk (#4695), pY416-Src (#6943) were purchased from Cell Signaling Technology, Inc. Antibodies against Flag (ab1162) was purchased from Abcam plc. Antibody against -Actin (TA-09) was purchased from ZSGB-BIO. Antibody against OspF is a generous gift from Prof. Feng Shao (NIBS, China). HRP-linked secondary antibodies were purchased from CST. PMA was purchased from Promega. Alkyne-Cy5 dye was purchased from Click Chemistry Tools (Scottsdale, AZ, USA). Protein purifications were performed on an AKTA START protein purification system (GE Healthcare). Western blot images were captured on a ChemiDoc XRS+ molecular imager (Bio-Rad) or Tanon 5200 Multi image system (Tanon). Luminescence of cell samples were acquired on a Synergy H4 microplate reader (Bio-Tek). Confocal microscopy images were obtained on LSM 700 laser scanning confocal microscope (Zeiss). To a mixture of 1 (1.0 eq., 2 mmol, 630 mg), Fmoc-lysine (HCl) (1.2 eq., 2.4 mmol, 972 mg) and NaHCO 3 (3.0 eq., 6 mmol, 504 mg) in a 25 mL flask, 4 mL DMF was added. The mixture was stirred at room temperature for 8 hours. After completion, the solvent was evaporated under reduced pressure. And the crude product was purified by flash column chromatography on silica gel using DCM and MeOH as eluent, followed by second flash column chromatography on silica gel using EtOAc and hexanes as eluent. 511 mg (47% yield) of Fmoc-PABK was obtained as colorless oil. 1  Fmoc-PABK (0.5 mmol, 260 mg) was dissolved in DCM (8 mL), piperidine (1.6 mL) was added slowly at room temperature. The mixture was stirred for 1 hour. The volatile materials were evaporated under reduced pressure, and the residue was purified by flash column chromatography on silica gel using DCM and MeOH as eluent. For using in cells, the product was purified again using reverse phase HPLC (eluent = MeCN/H 2 O, gradually changing from 5/95 to 100/0 in 20 minutes). After solvent removal under reduced pressure, the residue was then dissolved in 1 mL MeOH with 10 L concentrated HCl, and precipitated by adding Et 2 O. The solid was collected and washed with MeCN (3 × 1 mL). The product was obtained as a pale yellow solid, 35 mg (20% yield). 1

Expression and purification of proteins containing PABK in E. coli
The plasmids pSupAR-Mb-DiZPK-RS and pBAD-GFP-N150TAG-His 6 (or pBAD-HdeA-V58TAG-His 6 ) were co-transfected into E. coli DH10B cells. Cells were recovered in 1 ml LB medium for 1 h at 37 °C before further growth in LB medium containing ampicillin (50 mg/ml) and chloramphenicol (34 mg/ml) overnight at 37 °C. After 1:100 dilutions in 100 ml LB medium containing ampicillin (50 mg/ml) and chloramphenicol (34 mg/ml), the cell culture was grown at 37 °C to an OD 600 ~0.6, at which point 1mM PABK was added to the culture (final concentration). The bacteria were grown at 37 °C for 30 min before being transferred to 30 °C for induction in the presence of 0.02% arabinose for 12 h. The bacteria were collected by centrifugation (4500 rpm, 10 min) followed by resuspension in lysis buffer (20 mM Tris-HCl, pH 7.8, 500 mM NaCl). Lysate after sonication was loaded onto a Ni-NTA column (Histrap 5 ml, GE Healthcare), which was washed with 30 ml washing buffer (20 mM Tris-HCl, pH 7.8, 500 mM NaCl, 40 mM imidazole) and then eluted with elution buffer (20 mM Tris-HCl, pH 7.8, 500 mM NaCl and 250 mM imidazole) to yield target protein carrying PABK. Target proteins were then desalted into DPBS.

Expression and photocrosslinking of PABK-incorporated HdeA
Plasmids pSupAR-Mb-DiZPK-RS and pBAD-HdeA-V58TAG-His 6 or pBAD-HdeA-F35TAG-His 6 were co-transfected into E. coli DH10B cells. HdeA expression was described above. 1 ml of the cultures were treated by acid-stress buffer (10 mM Sodium Citrate, 150 mM NaCl, pH = 2) for 20 min then transferred into 24-wells. For pH 7 condition, 1 ml of the cultures were resuspended in PBS buffer. Photocrosslinking was performed by irradiation of the bacterial cells with UV light (365 nm) for 10 min using a Hoefer UVC 500 Crosslinker. The cells were collected by centrifugation then analyzed by SDS-PAGE and immunoblotting. 4

Labeling of PABK-incorporated EGFR on the cell surface and fluorescence imaging
Plasmids pEGFP-N1-EGFR-N128TAG and pCMV-MbPylRS/tRNAPyl CUA were cotransfected via X-tremeGENE HP (Roche) in DMEM (10% FBS) supplemented with 1 mM PABK. HEK293T cells then grew for additional 24 hours to express EGFR bearing PABK. Cells were then washed three times by DPBS to remove medium and free PABK, then reacted with Alkyne-Cy5 (50 M) in the presence of CuSO 4 (50 M), BTTAA (300 M), and sodium ascorbate (2.5 mM) in DPBS for 5 min at room temperature (RT). The reaction was quenched by the addition of BCS (500 M). The cells were washed three times by DPBS again and fixed with 4% PFA before fluorescence imaging. Cell images were captured by an LSM 700 laser scanning confocal microscope (Zeiss) equipped with a 63× oil immersion objective lens. EGFP and Cy5 channel was chosen with corresponding parameters. 5

Expression and purification of PABK-incorporated GFP in HEK293T cells
For GFP-Y40PABK expression in HEK293T cells, plasmids pCDNA4-GFP-Y40TAG-Flag and pCMV-MbPylRS/tRNAPyl CUA were co-transfected via X-tremeGENE HP (Roche) in DMEM (10% FBS) supplemented with 1 mM PABK. Cells then grew for additional 24 hours to express the desired protein bearing PABK at Y40 residue. Cells were collected by centrifugation (1000×g, 5 min) at 4 °C then resuspended in PBS buffer. Lysate after sonication was centrifuged at 12000 rpm for 5 minutes at 4 °C. Supernatants were incubated with ANTI-FLAG ® M2 Magnetic Beads (Sigma-Aldrich) according to its procedures for purification of GFP-Y40PABK(Flag). For analysis of decaging efficiency on GFP-Y40PABK(Flag), supernatants mentioned above were incubated with 500 M 4ax at 37 °C for 4 h, followed by purification procedures.

ESI-MS analysis of PABK-incorporated GFP
LC-MS analysis of GFP-Y40PABK(Flag) was performed using a Waters ACQUITY UPLC I-Class SQD 2 MS spectrometer with electrospray ionization (ESI). 0.1% formic acid in H 2 O as buffer A and 0.1% formic acid in acetonitrile as buffer B were used as the solvent system. LC separation for protein sample was carried out with a BEH300 C4 Acquity column (1.7 m, 2.1 × 100 mm), and positive mode was chosen for ESI-MS to analyze all samples. The total mass of proteins was calculated using MassLynx V4.1 software (Waters). Theoretical mass of the wild-type protein was calculated using Peptide Mass Calculator (http://www.peptidesynthetics.co.uk/tools/), and the theoretical mass for all modified proteins was adjusted manually.

Activation of PAB-caged fLuc in living cells
For PABK-incorporated fLuc (PfLuc) expression in HEK293T cells seeded in a 24-well corning plate, plasmids of pCDNA4-Luc-K529TAG-myc and PylRS-9/tRNAPyl CUA were cotransfected via X-tremeGENE HP (Roche) in DMEM (10% FBS) supplemented with 1 mM PABK. Cells expressing PfLuc were treated with 4ax in different conditions to undergo the strainpromoted 1,3-dipolar cycloaddition reaction after 24 h transfection and expression. Cells were collected and lysed by 50 L 1× Universal lysis buffer. 20 L cell lysate were mixed with 80 L luciferin solution (containing 20 mM Tricine, 1.0 mM MgSO 4 , 0.1 mM EDTA, 270 M coenzyme A, 500 M D-luciferin and 530 M ATP, pH 7.8) in a 96-well black plate. Relative bioluminescence unit was measured by Synergy 4 Hybrid Microplate Reader with integration time 5s and sensitivity at 120. Then bioluminescence images were taken by ChemiDoc using chemiluminescent channel. The left cell lysate was mixed with 5× loading buffer and boiled at 95 °C for 20 min. The crude sample was prepared for SDS-PAGE analysis to quantify the amount of the protein after further centrifugation.

4ax-induced OspF activation in vivo
For OspF-K134PABK expression in living cells, pCDNA3.1-Flag-HA-OspF-K134TAG and PylRS-9/tRNAPyl CUA were co-transfected by X-tremeGENE HP (Roche) in DMEM (10% FBS) supplemented with 1 mM PABK. HEK293T cells continued to grow for another 24 h. Cells were then incubated with 4ax at 500 M for 1.5 h. Then the culture medium was changed to fresh DMEM containing 5 M PMA to activate p-Erk for 20 min. Cells were then collected by centrifugation (1000×g, 5 min) and lysed by lysis buffer (40 mM Tris·HCl, pH 7.5, 500 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) containing protease inhibitor cocktail on ice for 30 min. Cell debris was discarded after centrifugation (13000 rpm, 5 min, 4 °C) and the supernatant was added with 5× loading buffer and boiled at 95 °C for 20 min. The sample was centrifuged at 13000 rpm for 10 min before being analyzed by 12% SDS-PAGE. Western blotting analysis was carried out to detect the full-length OspF protein as well as the dephosphorylation level of p-Erk by using antibodies against OspF, p-Erk1/2 and Erk1/2.

Cytotoxicity study of 4ax
HEK293T cells were seeded at a density of ~2,000-5,000 cells per well in flat-bottomed 96-well plates and continued to grow for 18 h. Cells were then incubated with different concentrations of 4ax for 3h and 72h. After incubation, each well's solution was replaced into the mixture of fresh DMEM and CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI) according to the manufacturer's instructions. The cell viability was determined by measuring the absorbance at 490 nm using a microplate reader. Absorbance results at 630 nm were used to subtract background.

Expression and activation of PABK incorporated Src in living cells
Plasmids of pCDNA4-Src-K295TAG-Y527F-myc and PylRS-9/tRNAPyl CUA were cotransfected via X-tremeGENE HP (Roche) in DMEM (10% FBS) supplemented with 1 mM PABK or CbzK. pCDNA4-Src-K295R-Y527F-myc, pCDNA4-Src-Y527F-myc and vector were transfected separately. After expression for 24 h, HEK293T cells were treated with 500 M 4ax for 3 h then collected for SDS-PAGE and immunoblotting analysis. Full length of Src and its autophosphorylation can be detected by antibodies against myc and pY416-Src separately.  Chemical decaging and ligation have been proceeded on various substrates. *: the reaction time and efficiency listed here only in accordance with the reaction conditions described in the manuscript.

Supplementary Schemes
Supplementary Scheme 1. Schematic illustration of functions of PABK incorporated protein of interest (POI). PABK incorporation into POI via genetic code expansion has versatile applications. PABK can undergo different chemistries, including bioorthogonal ligation (CuAAC), photochemistry for insertion or reduction, and decaging reaction with trans-cyclooctene (TCO). PABK also owns characteristic infrared band at about 2100 cm -1 (Fig. S16). for different time. The reactions were quenched by BCS and the labeling efficiency was determined by LC-MS. After 3 min, only 9.64% GFP-Y40PABK(Flag) remained and more than 90% proteins have been converted to the addition product (GFP-Y40PABK-Cy5). After 30 min, only the addition product can be observed, which demonstrated that the bio-conjugation reaction on PABK cannot lead to any cleavage process.