Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation

The use of living cells for the synthesis of pharmaceutical proteins, though state-of-the-art, is hindered by its lengthy process comprising of many steps that may affect the protein’s stability and activity. We aimed to integrate protein expression, purification, and bioconjugation in small volumes coupled with cell free protein synthesis for the target protein, ciliary neurotrophic factor. Split-intein mediated capture by use of capture peptides onto a solid surface was efficient at 89–93%. Proof-of-principle of light triggered release was compared to affinity chromatography (His6 fusion tag coupled with Ni-NTA). The latter was more efficient, but more time consuming. Light triggered release was clearly demonstrated. Moreover, we transferred biotin from the capture peptide to the target protein without further purification steps. Finally, the target protein was released in a buffer-volume and composition of our choice, omitting the need for protein concentration or changing the buffer. Split-intein mediated capture, protein trans splicing followed by light triggered release, and bioconjugation for proteins synthesized in cell free systems might be performed in an integrated workflow resulting in the fast production of the target protein.


Results
The following results are presented according to the workflow depicted in Fig. 1. However, several of the steps were also evaluated in together, in an integrated way.
Protein yields were determined from the western blots (WB; Fig. 2A,B) and are summarized in Table 1. Due to the limitations in the linear range of the WB-staining reagent the bands with highest concentrations were bleached out and therefore omitted from the quantitate analysis. The expression time in the BYL system was 16-20 hours at 25 °C, while hCNTF-NpuDnaE ΔC16 was expressed for 6 hours at 30 °C in the HeLa system. Overall, the amounts of hCNTF were 75% and 51% lower than the expected expression amounts of the positive controls of the BYL 17 and the HeLa systems respectively (as given by ThermoFisher Scientific, USA). Please note that due to the absence of β-mercaptoethanol in the loading buffer for the SDS-PAGE, previously purified hCNTF appears as 2 bands on the WB. The major band is the monomeric and the minor band the dimeric form. In previous work Itkonen et al. in our laboratory have optimized the expression and purification of hCNTF 18 . Here we can observe one band, the monomeric form of purified hCNTF, in a SDS-PGE gel with β-mercaptoethanol in the Laemmli buffer, stained with Coomassie brilliant blue.
Capture. The capture efficiency onto the beads in the CFPS matrixes was evaluated from the WBs before and after capture with peptides immobilized to magnetic beads from the relative amounts of hCNTF synthesized and remnant in the CFPS matrix ( Table 1). The amounts of hCNTF bound to the beads were 0.42 μg/mg beads and (B) Split intein mediated capture, for 3 hours. The capture peptide is either in solution or immobilized to the surface (the dashed line depicts both scenarios). In orange the 'Tag' can be any affinity tag. The red bar is a photo-cleavable amino acid, while the blue bar is an unnatural amino acid linked to a reactive moiety. At this stage the intein will splice itself out spontaneously, without co-factors 46,47 ; (C) After the intein reaction the CFPS matrix and side-products are washed away in under half an hour, and the final buffer is added (light blue); (D) after light triggered release (3 hours) POI is released into its final formulation. Bioconjugation can be performed now, later (for example in-vivo) or also at the end of step C before light treatment.
Purification. In order to validate split-intein mediated capture and protein trans splicing in solution, eGFP-NpuDnaE ΔC16 (enhanced green florescent protein-NpuDnaE ΔC16 ) was expressed in E. coli cells as a recombinant fusion protein. The crude, soluble fraction of the cell lysate was split and either incubated with peptide 1 or peptide 3 to test the feasibility of capture and release in a complex matrix with high protein background, in this case cell lysate. Western blotting was utilized to visualize the target protein with anti-polyhistidine antibody (Fig. 3A). The products eGFP-His 6 (hCNTF-(histidine) 6 ) and eGFP-λ-His 6 (hCNTF-photocleavable linker-(histidine) 6 ) were purified using affinity chromatography (Fig. 3B,C). The final product eGFP, prepared without a His 6 fusion tag, was clearly purified using affinity chromatography, demonstrating the feasibility of split-intein mediated capture and protein trans splicing in a complex matrix. The whole procedure, excluding the cloning experiments, was performed in 6 days.
The washing efficiency of captured and immobilized hCNTF from the Ni-NTA magnetic beads was determined in two scenarios: (a) split-intein mediated capture was performed in the BYL matrix follow by immobilization to Ni-NTA coated magnetic beads, and (b) split-intein mediated capture and protein trans splicing in the HeLa lysate matrix with prior immobilized peptides. Vigorous washing of the beads as instructed by the manufacture using the KingFisher (ThermoFisher Scientific) was equally effective for both scenarios and removed (50 ± 18) % of hCNTF from each previous washing step. Thus, before light triggered release, less than 1.5% of the remnant hCNTF was present. Vigorous washing with equal volumes are visualized in Fig. S4A (split-intein mediated capture and protein trans splicing with immobilized peptide in HeLa) and S4B (mock-split-intein mediated capture and mock-protein trans splicing in BYL). The washing steps depicted in Fig. 4 were less vigorous and concentrated before application to the SDS-PAGE gels in order to visualize if hCNTF was present in the washing step. After washing, hCNTF was cleaved off the beads via light triggered release (Fig. 4A,B) into the final buffer.

Bioconjugation.
To demonstrate proof-of-principle of moiety transfer from the capture peptide to hCNTF, we utilized peptides 5-7 where biotin was conjugated to the peptides prior to split-intein mediated capture and protein trans splicing. Firstly, we transferred biotin to hCNTF synthesized in cell free matrix with peptides 5-7 within the same matrix and observed a migration-shift in the SDS-PAGE gels (Fig. 5A) where the hCNTF-biotin appears to  The percentages are based on the relative volumes of the bands of hCNTF released from the magnetic beads compared to the total synthesized hCNTF in the same WB gels; c Immobilized peptide followed by split-intein mediated capture, washing, and light triggered release; d split-intein mediated capture and protein trans splicing in solution followed by immobilization, washing, and light triggered release; e corrected values are based on negative controls (Fig. S4). ND = Not Determined. be smaller, however, it migrates faster due its increased charge. This effect has been described earlier by Sano and Cantor (1990) 19 , where titration of streptavidin with biotin marked an increase in mobility on polyacrylamide gels. In the streptavidin-shift assay, hCNTF-biotin was detected, though the relative amount appears to be much lower than the difference of hCNTF before and after incubation with streptavidin (Fig. 5B). In Fig. 5B we observe once again the higher mobility of hCNTF-biotin in lane 4, and possibly in lane 6.
After background correction, 18% of hCNTF is liberated after the split-intein mediated capture and protein trans splicing, relative to the amount of hCNTF-NpuDnaE ΔC16 , with peptide 1 (Fig. 5B; lane 3), while peptide 5 is more efficient with 76% liberation of hCNTF. After incubation with streptavidin the relative amount of hCNTF in lane 5 did not change significantly (16%), while the amount of hCNTF-biotin in lane 6 compared to lane 4 is reduced by 56%. In comparison the amount of hCNTF-biotin-streptavidin, marked with an asterisk in lane 6, compared to the amount of hCNTF in lane 4 is 15%.
Light triggered release. In order to evaluate the cleavage efficiency of the capture peptide's photocleavablelinkers, the reduced peptides were incubated up to 30 minutes under UV light (365 nm; 12. mW) and analyzed with HPLC ( Table 2; Fig. S5). The main peak of the chromatogram after 0 min of UV-light exposure represents 100% of  non-cleaved peptide and its integrated peak area was compared to peaks at the same retention time of the same peptide. Peptide 1 and 5 do not contain a photocleavable moiety within the peptide structure, thus any observable decrease in the main peptide peak, and appearance of reaction product's peaks in the chromatogram are solely due to the photocleavable moieties. The most efficient photocleavable group evaluated in this study is 3-(amino)-3-(2-nitrophenyl)propanoic acid (ANP) 20 , while 4-|4-[1-(9-fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy]butanoic acid (CAS 162827-98-7) 21 is slightly more efficient than 2-nitrophenylalanine (F(2-NO 2 )) 22 .
We evaluated the photocleavage after (i) CFPS, (ii) split-intein mediated capture, and (iii) protein trans splicing in CFPS matrix followed by washing. Immobilization of hCNTF-λ-His 6 on magnetic beads was also evaluated. hCNTF-NpuDnaE ΔC16 alone was not affected by light treatment (Fig. S4A), while light treatment of the hCNTF-λ-His 6 liberated hCNTF (Figs 2A and 4). The release efficiency presented in Table 1 are conservative percentages. Due to technical limitations, the evaporation of the liquid from the mock-light triggered release samples (Fig. S4A), could not be compensated for, thus we overestimated the amount of hCNTF in the last lane of Fig. S4A of the negative control, resulting in a lower estimate of release of hCNTF in Figs 2A and 4. Hence, we can conclude that the release rates of hCNTF from the magnetic beads (5 mg beads/ml) is at least 6.3% if the peptide 3 is immobilized before split-intein mediated capture and protein trans splicing, while the release rate is at least 19% if immobilization occurs after split-intein mediated capture and protein trans splicing.

Discussion
In summary, we demonstrated proof-of-principle of a fast work-flow to prepare, purify and bioconjugate a pharmaceutical relevant hCNTF protein. Cell free expression of proteins is rapid with yields high enough if followed by sensitive screening methods. Here we evaluated a plant-based system tobacco plant BYL and a mammalian-based system based on HeLa cells. The higher yield in the HeLa CFPS system (49 mg/L of hCNTF), compared to BYL was unexpected, but this may be due to the stability of hCNTF. The expression in the HeLa system was done in 6 hours, while the recommended time of 16-20 hours was applied for the BYL system 23 . Initially storage in a 100 mM NaH 2 PO 4 , 50 mM NaCl, 2 mM DTT buffer (pH 8.0) following expression of hCNTF in E. coli 18 was not optimal, therefore a ThermoFluor assay 24 was performed. From the thermo-ramping profiles, it was evident that hCNTF unfolds at 42-48 °C (data not shown). For comparison, hCNTF expressed in E. coli at a level of >112 mg/L of total protein using a codon optimized gene 18 . The E. coli expression and purification was done in 5-6 days.
The capture efficiency of hCNTF-NpuDnaEN Δ16 by the capture peptides within the CFPS matrix differed for both systems, mainly due to non-specific binding of proteins already present. This is supported by the negative control (Fig. S4B) where we observe native protein binding and elution to peptide coated Ni-NTA, a known issue when using Ni-NTA surfaces 25 . The capture efficiencies were determined by comparing the relative amounts of hCNTF before and after capture. Split-intein mediated capture onto magnetic beads in CFPS matrix was highly effective up to 93%. The presence of other proteins, therefore, does not seem to limit the capture efficiency. The capture efficiency of the split intein used, in buffer, extrapolated from Fig. S15 from Shah et al. 26 appears to be 92-95%. In the same paper, the authors reported that the rate of the intein capture is lower at high salt concentrations. When compared to the specific binding capacities of His 6 -binding resins on magnetic beads, hCNTF is captured with both CFPS systems at a high to very high efficiency at similar bead concentrations 27 . However, the protein amounts bound via split-intein mediated capture followed by protein trans splicing are much lower than the capacity of the beads. In the BYL system we immobilized 0.42 μg hCNTF per mg of beads and for the HeLa system this was 1.14 μg hCNTF per mg of beads, while the binding capcity of the beads, according to the manufacturer is over 40 μg GFP per mg of beads. Since our relative binding efficiency is high, but the total amount of hCNTF is relatively low, it seems the maximal capacity of the magnetic beads functionalized with the capturepeptides was not reached.
In the EnBase TM cultivation E. coli follows a linear growth curve 28 , the total protein background in the E. coli lysate was in a similar range of 75-100 fg protein per cell previously reported for slow dividing cells 29 . As shown in Fig. 3, eGFP-NpuDnaE ΔC16 treated with peptide 1 and 3, resulting in eGFP-His 6 and eGFP-λ-His 6 , could be purified by use of Ni-NTA affinity chromatography. Therefore, we demonstrated that split-intein mediated capture and protein trans splicing in a complex and high protein background is possible in the crude, soluble fraction of E. coli lysate.
Following the capture and binding of hCNTF to Ni-NTA magnetic beads, 5 simple washing steps removed 98.5% of the remaining 7% of hCNTF. After demonstrating the photocleavability of the capture peptides (Table 2) with comparable values reported earlier [20][21][22] , we demonstrated their functionality in CFPS matrixes. Light triggered release was most effective when protein trans splicing was allowed in the complex CFPS matrix before immobilizing to magnetic beads where 19% of immobilized hCNTF was released from the beads due to light treatment. Compared to His 6 fusion tags, coupled with Ni-NTA resin on magnetic beads, their elution (or release) efficiency is between 5-94% for 4 mg beads/ml 27 . Most likely an overcrowded peptide surface is less optimal for a functionally folded intein to form after split-intein mediated capture. During validation experiments of an earlier study (data not show) we found that for flat chitin-SPR sensors approximately 20% of chitin binding domain fused to NpuDnaE ΔC16 was an optimal distribution of the capture peptide 30 . One other limitation of the use of magnetic beads for light triggered release, is the difficulty to penetrate the magnetic bead slurry in the plastic wells of the KingFisher strips (ThermoFisher Scientific, USA) from above with UV-light. Furthermore, though the beads were mixed in a microtiter plate shaker throughout the UV-light exposure in a closed-box, the beads did sediment.
In addition to split-intein mediated capture, protein trans splicing, and light triggered release as an alternative protein purification protocol, we also wanted to demonstrate an alternative post-translational modification via intein mediated moiety transfer as an integrated part of the workflow, because bioconjugation is an important aspect of (biological) drug development [31][32][33] . As shown in the cartoon in Fig. 1, after protein trans splicing the POI is covalently linked to the capture peptide. We demonstrated that biotin was transferred from the capture peptide onto hCNTF in one step. In this reaction, no byproducts of the conjugation reaction needed to be removed prior to bioconjugation to the protein 34 . Furthermore, it appears that most of the hCNTF has been bioconjugated (Fig. 5A). Peptide 5 was utilized in a traditional streptavidin-shift assay 35 , since its split-intein mediated capture and protein trans splicing was more effective than with peptides 6 and 7 (Fig. 5B). In this reaction approximately 56% of hCNTF was biotinylated, however only 15% of that amount bound to streptavidin. The main reason for this low percentage is the over 100-fold excess of peptide 5 in the reaction mixture. This 'captures' a significant amount of the streptavidin away, which in turn cannot bind to the biotinylated hCNTF. In addition, the human anti-hCNTF antibody used for visualization might be less specific for the hCNTF-biotin-streptavidin complex than for hCNTF and hCNTF-biotin respectively. However, even though optimization is needed, moiety transfer after split-intein mediated capture and protein trans splicing is clearly demonstrated with cell-free protein synthesized hCNTF and in CFPS matrix.
The current limitations of the workflow depend on the ability to express the POI-intein fusion as a soluble protein and the efficiency of light cleavage while the captured protein resides on magnetic beads. Another current limitation is the yield in absolute amounts. CFPS is scalable 16 and the amount captured is related to the total surface areas covered with the capture peptides. Even if protein production within a CFPS matrix is slower than expected, the CFPS reaction can be prolonged to capture the same amount over a longer period of time. Currently we are evaluating this new work-flow on flat surfaces to optimize the light triggered release efficiency. We will couple this work-flow with process analytical techniques to evaluate the amount and quality of the pharmaceutical protein produced as well as in-line assays relevant to its function. Finally, we will indent to scale up the capture surface to increase the yields.
All DNA fragments were separated on 1% agarose gels, and bands of the appropriate sizes excised and cleaned up with NucleoSpin® Gel and PCR Clean-up columns (Macherey-Nagel, DE). The purified DNA fragments were assembled into plasmids using NEBuilder® HiFi DNA Assembly Cloning Kit (NEB, MA, USA) and the yielded expression plasmids were transformed into NEB-5α (NEB, MA, USA) E. coli competent cells 36 . Multiple colonies were picked and screened for the insertion of the hCNTF-NpuDnaE ΔC16 sequence by colony PCR using 5′-TAATACGACTCACTATAGGG-3′ and 5′-ttttttttttttttttttttttCTAGCGCTCAACTCCAATGTC-3′, and 5′-ATGTAATACGACTCACTATAGAAA-3′ and 5′-AGGTCCAAACCAAACCA-3′ as forward and reverse primers for HeLa and BYL constructs, respectively. Positive clones were propagated and the sequences of the amplified plasmids verified by gene sequencing (GATC Biotech, DE). Verified plasmid constructs were further amplified and purified to high concentration and purity using NucleoBond® Xtra Midi columns (Macherey-Nagel, DE). Plasmids were then transformed separately to E. coli strain BL21(DE3) (Invitrogen, USA) for protein production and in parallel stored at −20 °C for CFPS.

Cellular protein expression and purification.
Expression and purification of His 6 -hCNTF used for the controls was performed as previously described 18 . Briefly summarized, His 6 -hCNTF was produced in Rosetta 2 (DE3) pLysS with the auto-inducing media TBONEX (450 ml in 2000 ml baffled flask; 25 °C at 270 rpm for 30 h). Frozen cell pellets were freeze thawed and resuspended (3x) in 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, 1 mM DTT, protease inhibitor cocktail, and lysonase (pH 8.0). His 6 -hCNTF present in the soluble cell lysate fraction was purified with a Ni-IDA resin and size exclusion chromatography. The protocol had one adaption: a ThermoFluor assay 24 was performed to find a buffer (buffer A) which provided a better His 6 -hCNTF stability SCiEnTiFiC RepoRTS | (2018) 8:11967 | DOI:10.1038/s41598-018-30435-4 than the original storage buffer. Expression of eGFP-His6-NpuDnaE ΔC16 was carried out in BL21(DE3) E. coli cells. First the transformants were grown o/n in LB Medium with 100 μg/ml ampicillin and 1% (w/v) glucose at 30 °C from glycerol stocks; 2 ml o/n culture was used to inoculate 50 ml of EnPressoB medium 28 with 100 µg/ml ampicillin at 30 °C; 225 rpm (1″ amplitude shaker) according to manufacturer's instructions in high yield flasks 37 . Cells were induced with 0.4 mM IPTG after 24 h and grown for another 24 h under the same conditions. The cells were separated from the media with centrifugation (16 000 * g for 15 min) and then lysed by freezing o/n at −20 °C in the presence of DNAse (2.5 U/ml), RNAse (2.5 U/ml), lysozyme (15 μg/ml), 0.1 mM PMSF (ThermoFisher Scientific, USA), and MgCl 2 in buffer A. The insoluble fraction was removed by centrifugation (16000 g for 45 min). The crude, soluble E. coli lysate was kept on ice until further use. After incubating the cell lysates with either peptide 1 or peptide 3 Table 3) for 3 hours, the products of protein trans splicing were purified with a Protino ® 96 Ni-NTA well plate with using the vacuum manifold (Macherey-Nagel, Germany) according to the manufactures instructions using buffer A and recommended imidazole concentrations. Different wash volumes were evaluated and performed (N = 6), elution was as instructed and was performed four-fold. The resulting protein fractions of the wash and eluate steps were visualized with SDS-PAGE gels using coomassie brilliant blue R-250 (ThermoFisher Scientific, USA) Cell free protein synthesis (CFPS). E. coli BL21(DE3) S12 and S30 lysates [38][39][40][41] , tobacco plant or BYL 17 , E. coli (Promega, USA, order number L1110), wheat germ (Biotechrabbit, Germany, order number BR1401001), and mammalian HeLa cells (ThermoFisher Scientific, USA, order number 88881) were utilized for protein expression according to the published protocols or manufacturer's instructions. The quality of each CFPS reaction was checked by use of a positive control protein (GFP) in parallel to the CFPS reactions of the samples. Expression levels were also compared to a negative control where no DNA was added to the reaction. Continuous-exchange cell-free expression (CECF) of Strep-Tag II _TEV-HspA1 was performed in wheat germ lysates. CFPS of GFP was performed in E. coli BL21(DE3) S12 and S30 lysates with the PremixPlus reaction mixture from the Promega E. coli kit and E. coli lysate (Promega, USA) with the PremixPlus, the 'Swartz' reaction mix 41 , and the 'EMBL' reaction mix 39 . CFPS of hCNTF-NpuDnaE ΔC16 was carried out in the BYL and HeLa systems. Briefly, required reaction components were mixed, plasmid DNA template was added and the reaction mixtures incubated accordingly; BYL CFPS reactions were incubated in an incubator-shaker (25 °C for 20 h) in 96-well plates while HeLa CFPS reactions took place at 30 °C during 6 h in 1.5 ml microcentrifuge tubes. The synthesized proteins in the CFPS matrix were used in subsequent experiments immediately.

Capture and Release. The validity of the intein-mediated protein trans splicing of the artificially split
NpuDnaE intein (NpuDnaE ΔC15 /NpuDnaE C15 42,43 ; see availability of data and materials) in the capture and release of expressed protein was studied. The expressed, captured and released hCNTF was studied with Western Blotting. To verify that protein trans splicing can indeed ligate a C-terminal peptide sequence with e.g. a photocleavable moiety and a His 6 -tag (for the capture and release) to the expressed POI, the following experiments were set up. PTS reactions between photocleavable peptide 2/peptide 3 (Table 3) and CFPS reactions containing expressed hCNTF-NpuDnaE C∆16 were carried out for 3 hours at room temperature. The resulting putative His 6 -tagged hCNTF was captured with a 1 mg of HisPur™ Ni-NTA Magnetic Beads, corresponding to a 5-10 mg/ ml final concentrations of beads (ThermoFisher Scientific, USA). All handling of the magnetic beads was carried out with the KingFisher™ Purification System (ThermoFisher Scientific, USA). The unbound proteins and components of the CFPS reactions were subsequently removed from the beads by five washing-steps with 100 μl 0.05% Tween-20 in PBS (pH 8.0). Release of hCNTF was carried out by photocleavage with UV light (365 nm; 12.5 mW) for 0-360 min.
The feasibility of utilizing the intein ligation reaction itself in the capture and subsequent release of the expressed protein was also studied. In this setup, peptide 3 was first immobilized on the Ni-NTA beads. Intein ligation reaction between immobilized peptide 3 and CFPS expressed hCNTF-NpuDnaE ΔC16 was carried out overnight in room temperature, resulting in the ligation product, hCNTF-His 6 , that remains immobilized on the magnetic beads. After removal of unbound proteins and CFPS reaction components by wash steps, release of hCNTF was carried out by photocleavage with UV light (365 nm; 12 mW) for 0-360 min.  To verify that the capture and release were indeed achieved via the intein ligation reaction, control experiments were carried out. To rule out nonspecific binding to the Ni-NTA beads, a mock binding experiment was carried out using only the CFPS expressed hCNTF-NpuDnaE ΔC16 with the beads. After removal of unbound proteins and components of the CFPS reaction by wash steps, 'release' was carried out by photocleavage with UV light (365 nm; 12.5 mW) for 0-360 min. To verify the release having taken place via cleavage of the photolabile unnatural amino acids, the non-photocleavable peptide 1 was first immobilized on the Ni-NTA beads. Intein ligation reaction between immobilized peptide 1 and CFPS expressed hCNTF-NpuDnaE ΔC16 was carried out overnight in room temperature, resulting in the ligation product, hCNTF-His 6 , staying immobilized on the magnetic beads. After removal of unbound proteins and components of the CFPS reaction by wash steps, 'release' of hCNTF was carried out by photocleavage with UV light (365 nm; 12.5 mW) for 0-360 min.
Bioconjugation. Protein trans splicing reactions were carried out between expressed hCNTF-NpuDnaE ΔC16 and NpuDnaE C16 peptide 1 or modified peptides 5, 6 or 7; the biotinylated peptide 5, peptide 6 and peptide 7 were used to assess if the respective modifications (Table 3) affect the Protein trans splicing e.g. through steric hindrance, while the non-conjugated peptide 1 was used as a positive control for the reaction. Briefly, hCNTF-NpuDnaE ΔC16 in HeLa CFPS reaction matrix was mixed with peptide 1, peptide 5, peptide 6 or peptide 7 (all 100 µM; reduced with 25 mM TCEP at 27 °C for 30 min) in buffer C and the PTS allowed to take place in room temperature for 2 h. Reactions were halted with the addition of 4X Laemmli sample buffer.
In a similar setup, peptide 5 was used to assess transferring biotin onto the protein of interest via PTS, while peptide 2 was used as a negative control. PTS reactions were set up as described earlier and carried out at room temperature overnight. Taking advantage of the well-documented strong and SDS-resistant binding between streptavidin and biotin, a streptavidin gel-shift assay (adapted from Fairhead and Howarth and Sorensen et al. 35,44 ) was used to confirm transfer of biotin moiety to hCNTF; the described PTS reaction setups yielding the putative non-biotinylated and biotinylated hCNTF products were mixed with streptavidin in PBS and the streptavidin-biotin binding allowed 3 hours in room temperature. Detection of hCNTF biotinylation and subsequent streptavidin binding was carried out by Western Blotting.

HPLC.
To study the photocleavage efficiency and rate of synthesized NpuDnaE C16 -peptides with varying linkers and conjugates, the peptide samples were dissolved in ultra-pure water and reduced with 25 mM TCEP at 27 °C for 30 min and then exposed to UV-light (365 nm; 12.5 mW) for 0-30 min. The generated peptide traces were separated with an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA) with UV detection at 216 nm. A Discovery® BIO Wide Pore C18 (150 × 4.6 mm, 5 µm; Sigma-Aldrich) column maintained at 25 °C and at a flow rate of 1.5 ml/min was used for all analyses. Samples (15 µl) were injected into the column using water (A) and ACN (B), both with 1% TFA, as eluents. The gradient program (0-14 min 1 → 40% B, 14-16 min 40 → 70% B, 16-16.1 min 70 → 1% B, followed by 14 min equilibrium at 1% B) was used to elute all peptide samples. Resulting chromatograms were examined and processed with ChemStation software A.08.03 (Agilent Technologies); decrease in the AUC of the respective main peptide peak was used as a surrogate to measure light triggered cleavage of the different peptides.
Western blotting. Protein samples were resolved on precast 4-20% Mini-Protean® TGX Stain-Free SDS-PAGE gels (Bio-Rad) with PageRuler™ Prestained Protein Ladder (ThermoFisher Scientific, USA) or Precision Plus Protein™ WesternC™ Standard (Bio-Rad, USA) used as molecular weight markers. Separated proteins were transferred onto 0.2 µm nitrocellulose membranes with the Trans-Blot® Turbo™ Transfer System (Bio-Rad) according to manufacturer's instructions and the membranes blocked in 2% bovine serum albumin (BSA) in Tris-buffered saline, 0.05% Tween 20 (TBS-T) for 2 h in room temperature or overnight in +4 °C. Proteins were detected in a sandwich reaction with 0.1-0.2 µg/ml rabbit anti-hCNTF polyclonal antibody (ThermoFisher Scientific, USA) and 0.1 µg/ml horseradish peroxidase-conjugated goat anti-rabbit IgG (Merck Sigma Aldrich, DE) as primary and secondary antibodies, respectively. Staining was carried out with Amersham™ ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, USA) and the chemiluminescence detected with ChemiDoc™ XRS+ imaging system (Bio-Rad, USA). The band intensities were quantified using with the Image Lab™ software (Bio-Rad, USA) 45 . In brief, we image the WB with UV light and subtract the background after inspecting each lane. Known hCNTF quantities were used to calculate the amount of hCNTF on the sample gels. Overexposed band were excluded from the quantitative analysis. Membranes were rinsed with TBS-T between incubations.
Availability of data and materials. The datasets supporting the conclusions of this article are included within the article and the supplementary data file.
The intein nomenclature is according to Aranko et al. 46 , however since our capture peptides are 16 amino acids long, we use NpuDnaE ΔC16 instead of NpuDnaE ΔC15 , however the split-site is the same.