Intracellular Application of an Asparaginyl Endopeptidase for Producing Recombinant Head-to-Tail Cyclic Proteins

Peptide backbone cyclization is commonly observed in nature and is increasingly applied to proteins and peptides to improve thermal and chemical stability and resistance to proteolytic enzymes and enhance biological activity. However, chemical synthesis of head-to-tail cyclic peptides and proteins is challenging, is often low yielding, and employs toxic and unsustainable reagents. Plant derived asparaginyl endopeptidases such as OaAEP1 have been employed to catalyze the head-to-tail cyclization of peptides in vitro, offering a safer and more sustainable alternative to chemical methods. However, while asparaginyl endopeptidases have been used in vitro and in native and transgenic plant species, they have never been used to generate recombinant cyclic proteins in live recombinant organisms outside of plants. Using dihydrofolate reductase as a proof of concept, we show that a truncated OaAEP1 variant C247A is functional in the Escherichia coli physiological environment and can therefore be coexpressed with a substrate protein to enable concomitant in situ cyclization. The bacterial system is ideal for cyclic protein production owing to the fast growth rate, durability, ease of use, and low cost. This streamlines cyclic protein production via a biocatalytic process with fast kinetics and minimal ligation scarring, while negating the need to purify the enzyme, substrate, and reaction mixtures individually. The resulting cyclic protein was characterized in vitro, demonstrating enhanced thermal stability compared to the corresponding linear protein without impacting enzyme activity. We anticipate this convenient method for generating cyclic peptides will have broad utility in a range of biochemical and chemical applications.


MRNGLHHHHHHASVRPLNCIVAVSQNMGIGKNGDLPWPPLRNEFKYFQRMTTTSSVEGKQNLVIMGRKTWFSI PEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQPELASKVDMVWAVGGSSVYQEAMNQPGHLRL FVTRIMQEFESDTFFPEIDLGKYKLLPEYPGVLSEVQEEKGIKYKFEVYEKKDPGRNGL
. Plasmid map, DNA and amino acid sequences of recombinant mDHFR substrate.The protein (amino acid sequence in orange) was appended with a short linker, His 6 tag (in blue) for purification and AEP recognition sites at the N and C termini (black, underlined).NGL sequence near the N-terminus enables OaAEP1 to remove the upstream amino acids, revealing the cognate N-terminal recognition sequence (GL) for OaAEP1-mediated cyclisation.

General Information
Custom oligonucleotides and the pACYC-duet1 plasmid vector were purchased from Merck Sigma Aldrich.All restriction enzymes were purchased from ThermoFisher Scientific.Electrocompetent E. coli, buffers for molecular cloning, nucleotide triphosphate, Q5® High-Fidelity DNA polymerase, and NEBuilder® HiFi DNA assembly master mix was purchased from New England Biolabs.DNA Sanger sequencing performed by Eurofins Genomics.

Molecular Cloning
Linear mDHFR -The plasmid pACYC-Duet1-mDHFR was used for preparing linear mDHFR by IPTG induction.It was constructed by amplifying the genes encoding for His 6 -mDHFR then subcloned between KpnI and NdeI restriction sites.The ligated product was transformed into E. coli strain DH5α.Sequences of all plasmids were confirmed by DNA Sanger sequencing.
Cyclic mDHFR -The plasmid pACYC-Duet1-OaAEP1-mDHFR was used for co-expression of OaAEP1-C247A and mDHFR by IPTG induction to prepare cyclic mDHFR.It was constructed by amplifying the gene encoding His 6 -Ubquitin-OaAEP1-C247A by PCR using primers ST14I-F and ST14I-R into the plasmid pACYC-Duet1-mDHFR linearized by PCR using primers ST14V-F and ST14V-R.The PCR products were digested by DpnI (ThermoFisher) then assembled by Gibson Assembly using NEBuilder® HiFi DNA assembly master mix and transformed into E. coli strain DH5α.Sequences of plasmids were confirmed by DNA Sanger sequencing.Primer ST14I-F GGAGATATACCATGGGCATGGCG Primer ST14I-R CGGCCGCAAGCTTTTAGTCGTTCGCC Primer ST14V-F AAGCTTGCGGCCGCATAATG Primer ST14V-R GCCCATGGTATATCTCCTTATTAAAG Recombinant expression and purification -Recombinant expression was performed in E. coli strain SHuffle T7 express.The cells were grown at 30 °C in LB medium until the OD 600 reached 0.7, at which point protein production was induced by adding IPTG (to a final concentration of 0.2, 0.5 or 1.0 mM).After culturing at 16 °C for 18 h, cells were harvested by centrifugation at 4000 g and 4 °C for 20 min.Cell pellets were stored at -20 °C until purification.The cell pellet from a 1 L culture was suspended in 40 mL of lysis buffer containing 25 mM Tris.HCl (pH 7.5), 500 mM NaCl, 20 mM Imidazole, 0.1 mg/mL lysozyme, DNase I and cOmpete protease inhibitor cocktail.After lysis by sonication and clearance by centrifugation (14000g, 4 °C, 15 min), the supernatant was loaded onto a 5 mL of HisTrap FF (Cytiva).The column was washed with 10 column volumes of lysis buffer, and the remaining bound protein was eluted over a linear gradient from 20 to 500 mM imidazole over 20 column volumes.Fractions containing the desired protein were combined and concentrated using an Amicon Ultra-15 centrifugal filter unit (10 kDa MWCO) (Merck Millipore), then further purified by size exclusion chromatography (Superdex 75 pg 16/600, GE Healthcare) that had been preequilibrated in 25 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM DTT. Fractions containing desired protein were combined, concentrated using an Amicon Ultra-15 centrifugal filter unit (10 kDa MWCO) (Merck Millipore) then stored at -80 °C.
Mass Spectrometry (LC-MS) -LC-MS analyses were performed using an Agilent QTOF 6545 with Jetstream ESI spray source coupled to an Agilent 1260 Infinity II Quat pump HPLC with 1260 autosampler, column oven compartment and variable wavelength detector (VWD).The MS was operated in positive ionization mode (gas temperature at 350°C, drying gas flow at 11 L min -1 , nebulizer gas flow at 50 psi, sheath gas temperature at 400°C, sheath gas flow at 12 L min -1 ).The VCap, Nozzle, Fragmentor and Skimmer voltages were set to 4000, 1000, 200 and 65 V respectively.The chromatographic separation (2 µL sample injection) was performed on an Agilent polymeric reversed phase (PLRP-S, 2.1 x 50 mm, 3 µm, 300 Å) at 0.2 mL/min using mobile phase A (H 2 O with 0.1% formic acid), and B (acetonitrile with 0.1% formic acid).Gradient elution started at 5% B, changed to 50% B at 8 min, then to 90% B at 9 min, held at 90% B until 10 min, then returned to 5% B for re-equilibration in a total 13 min run.Data analyses were performed in MassHunter BioConfirm 10.0.
Analytical Size Exclusion Chromatography -Analytical size exclusion chromatography was performed by injecting 100 µL of protein sample on a Superdex 75 Increase 10/300 GL (GE Healthcare) with 25 mM Tris-HCl (pH 7.5), 100 mM NaCl flowing at 0.8 mL min -1 on an AKTA FPLC system.Elution and therefore retention time was monitored by measuring the absorbance at 280 nm wavelength.

Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI-TOF MS)
-To co-crystallise the mDHFR reaction mixture for MALDI-TOF analysis, a ground steel well plate was wetted with 0.5 µL matrix (Sinapic acid, 10 mg mL -1 ), followed by 0.5 µL of reaction mixture, then 0.5 µL matrix.The sample was allowed to dry in ambient conditions before analysis.MALDI-TOF MS spectra were recorded on a Shimadzu Axima iD Plus Performance MALDI-TOF TOF spectrometer fitted with a 6mW laser operating at 337 nm recording in linear positive mode.
Chemical modification of protein N-terminus -2-pyridinecarboxyaldehyde (2-PCA) was purchase from Merck.N-terminal protein modification was performed according to reported protocol. 1Briefly, mDHFR (linear or cyclic) was incubated with 2-PCA in 25 mM sodium phosphate buffer (pH 7.5) at a final concentrate of 15 µM mDHFR and 2 mM 2-PCA.The reaction mixture was incubated at room temperature (20 °C) overnight (19 h).The reaction mixtures were analysed by MALDI-TOF MS Circular Dichroism (CD) -CD measurements were taken on an Applied Photophysics Chirascan with 200 µL samples in 1 mm path-length CD cells.Protein samples were diluted in 20 mM potassium phosphate (pH 7.4), 100 mM potassium fluoride.For full spectra, three scans between 190 and 300 nm were collected with a bandwidth of 1 nm and data sampled at a rate of 0.5 s -1 .These scans were averaged, a blank was subtracted, and the values were converted to molar residue ellipticity (MRE).Thermal denaturation experiments were performed by measuring the ellipticity at 209 nm over a temperature gradient from 4 to 90°C at 1°C increments.Post-melt scans at 20°C confirmed the proteins were irreversibly denatured as they did not overlay with the pre-melt scans.To determine the melting temperature (T m ), the thermal denaturation curves were converted to MRE and fitted to the Boltzmann sigmoidal equation using OriginPro graphing and analysis software.To estimate the fraction of unfolded protein during thermal denaturation, lower and upper bounds of each data set were linearly extrapolated using the 10 data points at 4 -13°C and 76 -85°C, respectively.The MRE from each sample was then fitted to respective lower and upper bounds using the following equation:

𝑀𝑅𝐸 -𝐿𝑜𝑤𝑒𝑟 𝑏𝑜𝑢𝑛𝑑 𝑈𝑝𝑝𝑒𝑟 𝑏𝑜𝑢𝑛𝑑 -𝐿𝑜𝑤𝑒𝑟 𝑏𝑜𝑢𝑛𝑑
The fraction of unfolded protein was plotted against temperature to present normalized thermal denaturation profiles of linear and cyclic mDHFR.DHFR activity assay -A colorimetric assay kit (Merck CS0340) was used to measure the activity of purified mDHFR enzymes.Cyclic or Linear-mDHFR (50 nM) and NADPH (60 µM) were mixed in assay buffer.For the blank reaction, a volume of assay buffer was added instead of enzyme (Fig S4).Reactions were initiated by the addition of DHF (50 µM) and the absorbance at 340 nm of samples was measured using a 10 mm pathlength quartz cuvette in a Varian Cary 50 UV-Vis spectrophotometer.The specific activity was calculated using the following equation:

Figure S2 .
Figure S2.Plasmid map, DNA and amino acid sequences of recombinant OaAEP1-C247A employed for the production of cyclic mDHFR.Residues 24-328 of OaAEP1 (black, underlined) was employed with a C247A mutation.The enzyme was appended with a His 6 tag (in blue) to enable purification and a ubiquitin fusion (in orange) to enhance solubility.

Figure S3 .
Figure S3.SDS PAGE analysis of the protein contents in the host cell upon IPTG-induced gene expressionusing different concentrations of IPTG (0.2, 0.5, 1.0 mM final concentration).