4-azido-L-phenylalanine

Abstract

Vibrio natriegens is the fastest growing organism identified so far. The minimum doubling time of only 9.4 min, the ability to utilize over 60 different carbon sources and its non-pathogenic properties make it an interesting alternative to E. coli as a new production host for recombinant proteins. We investigated the ability of the engineered V. natriegens strain, Vmax™ Express, to incorporate the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into recombinant proteins for NMR applications. AzF was incorporated into enhanced yellow fluorescent protein (EYFP) and MlaC, an intermembrane transport protein, by stop codon suppression. AzF incorporation into EYFP resulted in an improved suppression efficiency (SE) of up to 35.5 ± 0.8% and a protein titer of 26.7 ± 0.7 mg/L. The expression levels of MlaC-AzF even exceeded those of E. coli BL21 cells. For the recording of 1H-15N and 19F NMR spectra, EYFP-AzF was expressed and isotopically labeled in minimal medium and the newly introduced azido-group was used as coupling site for NMR sensitive 19F-tags. Our findings show that Vmax is a flexible expression host, suitable for the incorporation of ncAAs in recombinant proteins with the potential to surpass protein yields of E. coli. The presented method suggests the implementation of V. natriegens for expression of isotopically labeled proteins containing ncAAs, which can be chemically modified for the application in protein-observed 19F-NMR.

Introduction

In this work, we demonstrate SE of up to 35.5 ± 0.8% obtained in Vmax. Plasmids designed for and commonly used in E. coli were transferred to the engineered V. natriegens strain Vmax™ Express for successful expression of recombinant proteins containing p-azido-L-phenylalanine (AzF) (Scheme A). Additionally, the newly introduced azido-group was used as coupling site for 19F-containing tags, applicable in NMR studies.Scheme 1A Structure of p-azido-L-phenylalanine. B Work flow for EYFP expression: Co-transformation of plasmids into E. coli BL21(DE3) and V. natriegens Vmax™ Express cells, selection of variants by a 96-well-plate screening, shaking flask expressions with best variants

Abstract

Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

Here, we focused on three light-sensitive Orai1 mutants, specifically Orai1 A137Bpa (TM2), Orai1 L174Bpa (TM3) and Orai1 A254Azi (TM4), as they showed robust UV light-induced activation. Each mutant contained the photocrosslinking UAA in a distinct TM domain surrounding the pore-lining TM1. Ca2+ imaging experiments showed that Ca2+ levels increased upon the exchange from 0 mM to 2 mM Ca2+-containing extracellular solution in Orai1 A137Bpa and Orai1 A254Azi, but not Orai1 L174Bpa overexpressing cells. Subsequent application of a 10 s UV pulse significantly enhanced Ca2+ entry, while a second 30 s UV light pulse did not further increase cytosolic Ca2+ concentrations (Fig. ; Supplementary Fig. ).Fig. 2Photocrosslinking of Orai1 TM domains can lead to CRAC channel-like activation.Schemes show one Orai1 subunit representing either Orai1 A137Bpa, Orai1 L174Bpa or Orai1 A254Azi in the absence of STIM1. Stars indicate sites for UAA incorporation. Ca2+ imaging measurements of Orai1 A137Bpa (a), Orai1 L174Bpa (b) and Orai1 A254Azi (c) using R-GECO1.2. Intracellular Ca2+ levels, represented by the normalized intensity of R-GECO1.2 co-transfected with the above mentioned UAA-containing Orai1 mutants in HEK 293 cells, were monitored initially in 0 mM Ca2+ solution followed by a 2 mM Ca2+ solution. Under 2 mM Ca2+ solution conditions, UV light was applied for 10 s and 30 s, respectively. d–f Time courses of Ca2+ current-densities after whole-cell break-in of above mentioned UAA-containing Orai1 mutants. UV light was applied for 15 s during the recording leading to UV-induced activation of Ca2+ currents. g–i Corresponding current/voltage (I/V) relationships were taken after 100 s of measurement. Inlet represents reversal potential (Vrev) of wild-type CRAC channel (Orai1 + STIM1) versus light-sensitive Orai1 mutant currents. Single values are indicated in gray. Data represent mean values ± SEM of indicated number (n) of experiments. Mann–Whitney test was applied to show that Vrev are not significantly different. Detailed statistic values are shown in Supplementary Table . Source data are provided as a Source Data file.

Abstract

Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

Here, we focused on three light-sensitive Orai1 mutants, specifically Orai1 A137Bpa (TM2), Orai1 L174Bpa (TM3) and Orai1 A254Azi (TM4), as they showed robust UV light-induced activation. Each mutant contained the photocrosslinking UAA in a distinct TM domain surrounding the pore-lining TM1. Ca2+ imaging experiments showed that Ca2+ levels increased upon the exchange from 0 mM to 2 mM Ca2+-containing extracellular solution in Orai1 A137Bpa and Orai1 A254Azi, but not Orai1 L174Bpa overexpressing cells. Subsequent application of a 10 s UV pulse significantly enhanced Ca2+ entry, while a second 30 s UV light pulse did not further increase cytosolic Ca2+ concentrations (Fig. ; Supplementary Fig. ).Fig. 2Photocrosslinking of Orai1 TM domains can lead to CRAC channel-like activation.Schemes show one Orai1 subunit representing either Orai1 A137Bpa, Orai1 L174Bpa or Orai1 A254Azi in the absence of STIM1. Stars indicate sites for UAA incorporation. Ca2+ imaging measurements of Orai1 A137Bpa (a), Orai1 L174Bpa (b) and Orai1 A254Azi (c) using R-GECO1.2. Intracellular Ca2+ levels, represented by the normalized intensity of R-GECO1.2 co-transfected with the above mentioned UAA-containing Orai1 mutants in HEK 293 cells, were monitored initially in 0 mM Ca2+ solution followed by a 2 mM Ca2+ solution. Under 2 mM Ca2+ solution conditions, UV light was applied for 10 s and 30 s, respectively. d–f Time courses of Ca2+ current-densities after whole-cell break-in of above mentioned UAA-containing Orai1 mutants. UV light was applied for 15 s during the recording leading to UV-induced activation of Ca2+ currents. g–i Corresponding current/voltage (I/V) relationships were taken after 100 s of measurement. Inlet represents reversal potential (Vrev) of wild-type CRAC channel (Orai1 + STIM1) versus light-sensitive Orai1 mutant currents. Single values are indicated in gray. Data represent mean values ± SEM of indicated number (n) of experiments. Mann–Whitney test was applied to show that Vrev are not significantly different. Detailed statistic values are shown in Supplementary Table . Source data are provided as a Source Data file.

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

6 of the 20 positions containing Azi and 7 of the 20 positions containing Bpa led to constitutive activation prior to UV light irradiation, as monitored by R-GECO1.2. 7 mutants containing Azi and 7 mutants containing Bpa exhibited enhanced Ca2+ levels upon exposure to UV light - among those 4 with Azi and 4 with Bpa significantly. Interestingly, 4 positions containing Azi and 4 positions containing Bpa triggered constitutive activity in the non-excited state, which was partially reduced by UV light irradiation. All other Orai1 mutants containing UAA substitutions at distinct positions showed no significant Ca2+ influx neither before nor after UV light irradiation like Orai1 wild-type (Fig. , Supplementary Fig. ).

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

6 of the 20 positions containing Azi and 7 of the 20 positions containing Bpa led to constitutive activation prior to UV light irradiation, as monitored by R-GECO1.2. 7 mutants containing Azi and 7 mutants containing Bpa exhibited enhanced Ca2+ levels upon exposure to UV light - among those 4 with Azi and 4 with Bpa significantly. Interestingly, 4 positions containing Azi and 4 positions containing Bpa triggered constitutive activity in the non-excited state, which was partially reduced by UV light irradiation. All other Orai1 mutants containing UAA substitutions at distinct positions showed no significant Ca2+ influx neither before nor after UV light irradiation like Orai1 wild-type (Fig. , Supplementary Fig. ).

Insertion of photocrosslinking UAAs in TM2, TM3 and TM4 enables Orai1 activation upon UV light irradiation

In control experiments, we ensured that an intact plasma membrane expression of the mutants could only be achieved in the presence of the UAA and the expression of the respective tRNA/aaRS pair. In the absence of the latter, only weak background fluorescence was detectable in the cytosol, but not in the plasma membrane (Supplementary Fig. ) in line with abolished UV-mediated Ca2+ influx comparable to untransfected cells (Supplementary Fig. ). Furthermore, examination of the UV-evoked Ca2+ currents of cells co-expressing Orai1 A137Bpa-YFP and Orai1-CFP wild-type as a function of the Orai1 A137Bpa:Orai1 fluorescence intensity ratio showed a tendency for higher maximum currents with increasing ratio and highest UV-induced activation when only Orai1 A137Bpa was expressed (Supplementary Fig. ), indicating efficient UAA incorporation. Moreover, rapid UV-mediated activation occured independent of endogenous STIM and Orai isoforms, as shown in CRISPR/Cas9 STIM1/STIM2 double-knockout (DKO), CRISPR/Cas9 STIM1/Orai1 DKO and CRISPR/Cas9 Orai1/Orai2/Orai3 triple KO (TKO) cells (Supplementary Fig. ), although to slightly lower maximum current levels, likely owing to lower expression levels in the KO compared to wild-type HEK293 cells (Supplementary Fig. ). Moreover, we showed that ER Ca2+ levels detected by LAR-GECO1 in HEK293 cells expressing either Orai1 or Orai1 A137Bpa compared to untransfected cells remained comparable before and after UV light irradiation and subsequent thapsigargin (TG)-induced store-depletion led to a similar decrease in Ca2+ levels (Supplementary Fig. ), thus excluding that UV light-triggered Ca2+ entry leads to Ca2+ overload in the ER.

Abstract

Gene therapy offers potentially transformative strategies for major human diseases. However, one of the key challenges in gene therapy is developing an effective strategy that could deliver genes into the specific tissue. Here, we report a novel virus-like nanoparticle, the bioorthgonal engineered virus-like recombinant biosome (reBiosome), for efficient gene therapies of cancer and inflammatory diseases. The mutant virus-like biosome (mBiosome) is first prepared by site-specific codon mutation for displaying 4-azido-L-phenylalanine on vesicular stomatitis virus glycoprotein of eBiosome at a rational site, and the reBiosome is then prepared by clicking weak acid-responsive hydrophilic polymer onto the mBiosome via bioorthogonal chemistry. The results show that the reBiosome exhibits reduced virus-like immunogenicity, prolonged blood circulation time and enhanced gene delivery efficiency to weakly acidic foci (like tumor and arthritic tissue). Furthermore, reBiosome demonstrates robust therapeutic efficacy in breast cancer and arthritis by delivering gene editing and silencing systems, respectively. In conclusion, this study develops a universal, safe and efficient platform for gene therapies for cancer and inflammatory diseases.

Site-Specific Codon Mutated Virus-Like mBiosome and Its Gene Transfection Efficiency

To prepare the mutated VSV-G enveloped biosome packaging the GFP mRNA (mBiosomeGFP), a series of steps were undertaken. Firstly, HEK 293T cells were co-transfected with the mutated pCMV-VSV-G plasmid along with other three plasmids, pIRE4-Azi, pCMV-capsid10-flag (encoding the capsid10 protein), and pCMV-cargoGFP (encoding the GFP mRNA). This transfection process was carried out in the presence of 1 mM Azi, as depicted in Fig. c. After the concentration of mBiosomes, the membrane proteins were extracted and analyzed by Western blot assay. The results clearly demonstrated a successful incorporation of mutated VSV-G proteins into the envelope of mBiosomes (Fig. d). In order to gain further insights, the morphologies, particle sizes and Zeta potentials of the mBiosomes were examined using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The TEM images showed that eBiosome had a spherical shape and was encapsulated with spike proteins, closely resembling general enveloped virus. Similarly, three different types of mBiosomes displayed similar morphological characteristics to the eBiosome (Fig. e). Moreover, the particle sizes and Zeta potential values of the three different types of mBiosomesGFP were evaluated and compared to the eBiosomeGFP. The results obtained from DLS analysis demonstrated that all three types of mBiosomesGFP shared comparable particle sizes and Zeta potential values with the eBiosomeGFP (Figs. f and S3).