Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system

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Plasmid construction

Sequence of the affinity clamp (PDZ-fibronectin fusion protein) was extracted from the PDB file 3CH8. Sequences of the anti-HSA scFv and Fab antibody fragments were derived from the PDB file 5FUO or from the US_2014_0186365 patent (sequence 4D2). Sequences of the anti-IL-6 scFv, anti-IL-6 Fab, anti-IFNα-1b scFv, and anti-IFNα-2a Fab were derived from the following PDB files: PDB:4CNI, PDB:4ZS7, PDB:3UX9, PDB:4YPG, respectively. 

With minor exceptions, all coding sequences for peptides and proteins (Supplementary Table 3) were supplied as gBlock synthetic fragments by IDT (Integrated DNA Technologies), carrying ~30 bp vector-complementary flanks and subcloned via the Gibson Assembly method to the NcoI/NotI-opened pLTE (AddGene: 67044) or pOPINE (GenBank: EF372397.1) plasmids (Supplementary Table 3) according to the manufacturer’s instructions (New England Biolabs). To create SFTI ORF with unique AGG and AGT codons to be reassigned to the encoding of unnatural amino acids, two cysteine codons in wtSFTI ORF were replaced with the respective AGG and AGT triplets using Q5® Site-Directed Mutagenesis Kit (NEB) in two successive PCR rounds with oligonucleotides shown in Supplementary Table 6.

Peptide quantification by AC-assay

The peptide quantification assay based on activation of allosterically regulated TVMV protease in the context of peptide biosensor was established earlier in our lab39. Briefly, a clamping of C-terminal RGSIDTWV between ePDZ and Fn3 domains of affinity clamp results in dislodgment of the inhibitory peptide from the active site of TVMV, leading to protease activation and subsequent cleavage of the otherwise quenched reporter substrate. Typically, the affinity-clamp assay was carried out in a buffer containing 50 mM Tris-HCl, 1 M NaCl, 1 mM DTT, and 0.5 mM EDTA (pH 8.0), supplemented with 1 μM of peptide biosensor and 15 μM of TVMV-substrate peptide. The reaction progress was monitored by exciting the sample at 330 nm and recording the fluorescence changes at 405 nm for 3 h using the BioTek Synergy 4 Multi-Mode Microplate reader.

Trypsin-inhibitory assay

Trypsin was freshly prepared at 400 pM in the assay buffer containing 0.1 M NaCl, 10 mM CaCl2, 0.005% Triton X-100, 0.1 M Tris-HCl pH 8.0. Trypsin at 100 pM was pre-incubated with peptide (twofold dilution series from 4 nM to 3.9 pM) in 200 μL of the assay buffer for 3 h at room temperature. After incubation residual trypsin activity was measured by adding the quenched fluorescent peptide substrate Boc-Q-A-R- 7-amido-4-methyl-coumarin to a final concentration of 10 μM. The fluorescence increase upon the release of 7-amido-4-methyl-coumarin was monitored on Infinite M1000 Pro plate reader (Tecan) every 30 s using excitation and emission wavelengths of 360 and 460 nm, respectively. To calculate IC50, the initial velocities of the substrate hydrolysis by trypsin in the presence of different concentrations of peptide were fitted to a nonlinear regression curve using the software package Prism (GraphPad Software) followed by determining the corresponding Ki value using a tight-binding equation66 and a Km value of 12 μM.

RGS-tag removal with trypsin and asparagine endopeptidase (AEP)

Agarose-immobilized trypsin (treated with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) to reduce the chymotrypsin activity) was purchased from ThermoFisher (#20230) and equilibrated with digestion buffer (0.1 M NH4HCO3, pH 8.0), aliquoted and stored at 4 °C as 50% slurry (vol/vol). 20 µL of mixed trypsin–resin suspension was transferred into the filter-bottom tube, spun at 5000 × g for 2 min and then mixed with SFTI-RGS peptide in digestion buffer containing 0.1 M NH4HCO3 buffer, pH 8.0 at 1:1 or 1:10 protease to peptide ratios followed by incubation for 3 h at 37 °C using the ThermoMixer (Eppendorf). The cleavage reaction was stopped by the addition of trifluoroacetic acid to 2.5% and vigorous shaking. Following quantification of prmSFTI by LC/MS using the calibration curve, the cleavage reaction was lyophilized and the peptide was resuspended in the appropriate volume of the assay buffer. For asparagine endopeptidase-mediated cleavage/cyclization of the reduced peptide, substrates containing the AEP-recognition motif were incubated at ≤280 µM with 12 µg/mL of OaAEP1b in the digestion buffer (50 mM CH3COONa, 50 mM NaCl, 1 mM EDTA, pH 5.0) overnight at room temperature.

RGS-tag removal with thrombin, carboxypeptidase A and Y, PreScission protease and hydroxylamine

Thrombin cleavage was performed using 20 pmol of RGS-fusion peptide and 2 NIH units of thrombin (Sigma-Aldrich #T7009) in a thrombin cleavage buffer containing 50 mM Tris-HCl, 10 mM CaCl2, pH 8.4 at 37 °C for 6–12 h.

Carboxypeptidase A was used in the resin-immobilized form (Sigma-Aldrich #C1261). 40 µL of 50% (~0.2 U) suspension was transferred to the filter-bottom tube and equilibrated with the cleavage buffer. Carboxypeptidase A cleavage was performed on 20 pmol of RGS-fusion peptide in a cleavage buffer containing 25 mM Tris-HCl, 500 mM NaCl, pH 7.5 at 37 °C for 6–16 h.

Yeast carboxypeptidase Y powder (Sigma-Aldrich, #C3888) was adjusted to 0.2 mg/mL with the cleavage buffer containing 50 mM Na3C6H5O7, pH 6.0. The cleavage reaction containing 2 pmol/µL of RGS-fusion peptide, 40 µg/mL of enzyme in the cleavage buffer was incubated at 37 °C for 0.5 h or overnight.

PreScission protease was expressed in E. coli and purified to homogeneity; the protease was adjusted to 4 mg/mL with storage buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, 1 mM DTT, pH 8.0, 20% glycerol). The cleavage of resin immobilized protein/ligand complexes derived from 100 µL of resin-assisted translation reaction (~20 µL of settled AC-coated resin gel) was performed in 20 µL of buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 20 µg/mL of PreScission protease at 4 °C for 16 h.

All cleavage reactions were terminated by the addition of an equal volume of 2.5% TFA. Sample aliquots withdrawn at different time points were purified using Pierce C18 Spin Tips (ThermoFisher) and concentrated by SpeedVac vacuum concentrator before analyzing by MALDI or LC-MS.

For hydroxylamine cleavage, AC-resin with 1–50 µg of bound peptide-RGS harboring accessible NG-cleavage motif was incubated at 45 °C for 4 h in cleavage buffer containing 2 M hydroxylamine in 15 mM Tris buffered to pH 9.3 with lithium hydroxide. After incubation, the flow-through containing cleaved peptide was collected by centrifugation at 3000×g and the beads were washed in six alternating washing steps with washing buffer (50 mM Tris-HCl, 0.5 M NaCl, 0.1% Triton X-100, pH 7.5) and water followed by three final washings with water. The remaining resin-bound peptide was eluted with 0.2% TFA.

Insecticidal activity assay

The insecticidal activity of the Dc1a and its cell-free produced analog was tested by microinjection in female Drosophila melanogaster fruit flies aged 3–4 days with an average weight of 0.8–0.9 mg according to the previously described method67. Briefly, the C-terminally unprocessed Dc1a (Dc1a-GTGSGG-RGSIDTWV) was dissolved in water for injection, and water was used as the respective negative control. The thrombin-cleaved Dc1a sample (Dc1a-GTGSGG-R) contained thrombin and the respective amount of thrombin was added to the control sample. The injection volume was 50 nL and three repeats of seven doses (each in n = 8 fruit flies) were used for each Dc1a analog. Female Drosophila were cooled on ice before injection. At 24 h after the injection, the fruit flies were monitored for lethal effects and the median lethal dose (LD50) was determined. The number of control flies affected at the respective post-injection time was subtracted from the number of affected flies. The corrected numbers of affected Drosophila flies were upscaled to 100% and the resulting percentages were used to interpolate the respective LD50 values by fitting log dose–response curves using nonlinear regression analysis.

Selective inactivation of specific endogenous tRNAs in E. coli S30 extract

For inactivation of selected tRNAs, the E. coli S30 cell lysate was incubated with M5-125 and R7 antisense oligonucleotides (Supplementary Table 4) at 37 °C for 5 min to sequester endogenous tRNASerGCU and tRNAArgCCU, respectively. Both oligonucleotides were added to 9 µM final concentration from 400 µM stock solutions. Following incubations, the lysate was chilled on ice, supplemented with protease inhibitor cocktail (cOmplete™ Protease Inhibitor Cocktail, Roche) from 50× stock to achieve 1.7× final concentration and left on ice for 15 min prior to in vitro translation.

Synthesis and purification of tRNAs

DNA templates containing T7-promoter- and tRNA-coding sequences were assembled in 3-step PCR reactions as described38 with minor modifications. Briefly, in step 1 forward oligonucleotide (F) containing T7-promoter sequence was combined with reverse oligonucleotide (R1) for overlap extension following two cycles of 1 min denaturation, 1 min 42 °C annealing and 30 s elongation. In step 2, 5′-GCGG-extended 3′-G-ending T7-promoter oligonucleotide was combined with R2-oligonucleotide spanning 3′-part of tRNA sequence for five amplification cycles with 1 min denaturing, 30 s 42 °C annealing and 15 s amplification. In step 3, both T7-promoter and R3-oligonucleotide complementary to 3′-part of tRNA were used at 7.5 µM concentrations for 28 amplification cycles with 30 s denaturing, 30 s annealing at 42 °C and 20 s elongation. After completion, PCR reactions were diluted 2.5-fold with water and the resulting PCR-products were purified by ethanol precipitation and dissolved in water. Run-off transcription using T7 RNA polymerase was performed for 3 h or overnight at 35 °C in a buffer containing 40 mM Hepes-KOH (pH 7.9), 20 mM Mg(OAc)2, 2 mM Spermidine, 40 mM DTT, 5 mM of each rNTP, 0.25 μM DNA template, 10 μg/mL T7 polymerase, and 0.25 U/mL yeast inorganic pyrophosphatase. The tRNA transcripts were purified by affinity chromatography using ethanolamine–Sepharose matrix as described previously38. Briefly, for 1 mL of transcription reaction, 0.2 mL of the settled matrix was used. Following 1 h incubation at 4 °C, the resin-bound tRNAs were extensively washed with a buffer, containing 200 mM NaOAc pH 5.2, 0.25 mM EDTA. tRNAs were eluted from the matrix into the buffer containing 2 M NaOAc pH 5.2, 10 mM MgCl2, 0.25 mM EDTA. tRNAs were ethanol precipitated and the pellets were dissolved in tRNA buffer containing 1 mM MgCl2 and 0.5 mM NaOAc (pH 5.0). The sequences of tRNAs and oligonucleotides are summarized in Supplementary Tables 5 and 7.

Purification of orthogonal aminoacyl-tRNA synthetases

The protein sequences of orthogonal tRNA synthetases are provided in Supplementary Table 6. Engineered tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii (AzFRS.2.t1) and pyrrolysine-tRNA-synthase from Methanosarcina barkeri (chPylRS) were expressed in BL21(DE3) RIL or Rosetta cells, respectively, and purified by Ni2+ affinity chromatography and gel filtration as described previously. Briefly, the protein expression was induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside at OD600 0.8 with the following overnight incubation at 20 °C. The cell pellet was resuspended in a binding buffer containing 50 mM sodium phosphate, pH 8.0, 0.3 M NaCl, 0.1 mM ATP, 5 mM 2-mercaptoethanol, and 20 mM Imidazole. Cells were disrupted using a continuous flow mode cell disruptor (Constant Systems) and proteins were purified on Ni2+ affinity chromatography. Following affinity-chromatography, AzFRS.2.t1 was further purified by gel filtration on a Superdex 200 column (GE Healthcare) in PBS. In the case of chPylRSAF N-terminal 6 his-tag was cleaved off by PreScission protease. The gel filtration was performed on Superdex 200 equilibrated with 40 mM HEPES-NaOH pH 7.9, 100 mM KCl, 10 mM MgCl2, 0.1 mM ATP, 1 mM TCEP, 10% (vol/vol) glycerol. Purified proteins were concentrated, snap-frozen in liquid nitrogen and stored at −80 °C in aliquots.

In vitro production of the macrocyclic peptide with unnatural bond

p-Azido-l-phenylalanine (AzF) and n-propargyl-l-lysine (PrK) were purchased from SynChem and Sirius Fine Chemicals, respectively. AC-resin assisted in vitro translation reaction was assembled with S30 lysate treated with the antisense oligonucleotides and additionally supplemented with 12 µM tRNAAzF4-CCU, 10 µM AzFRS.2.t1, 1.5 mM AzF, 15 µM tRNAPylO2-ACU, 30 µM chPylRSAF, 1.5 mM Prk. The reaction was initiated by the addition of SFTI(AGG, AGT)-RGS coding template (Supplementary Table 3). The reaction was incubated at 32 °C for 90 min with shaking at 1400 rpm. The resin was washed as described above, and the peptide was either subjected to cyclization via copper-catalyzed azide-alkyne cycloaddition (CuAAC) on-resin or off-resin following elution with 0.2% TFA. For on-resin CuAAC, the reaction volume was adjusted with water to the initial translation reaction volume and further mixed with 1/7 volume of 1 M potassium phosphate (pH 7.4) and the same volume of catalytic premix consisting of 1 mM CuSO4 and 5 mM of Cu+1-stabilizing reagent BTTP. The reaction mixture was gassed with nitrogen for at least 1 min and the cyclization was initiated by adding 1/14 volume of freshly prepared 100 mM sodium ascorbate solution (Sigma-Aldrich) followed by 1 h incubation at room temperature. For analytical LC-MS analysis, the resin was washed and the cyclized peptide was eluted with 0.2% TFA. The sample was cleaned up using Zip-tip (Pierce C18 Spin Tips) according to the manufacturer’s protocol and further eluted into 0.1% TFA, 80% ACN.

Reverse-transcriptase-coupled quantitative real-time PCR (RT-qPCR)

RT-qPCR using a previously selected pair of oligonucleotides targeting 16S rRNA was employed for comparative quantification of ribosomal content in bacterial S30 extract-based and reconstituted (PURE) translation systems. To avoid genomic DNA interference at the PCR stage, the respective translation reactions were treated with DNase I in 20 µL volume containing 4 U of DNase I (NEB, #M0303S), 4 µL of 50-fold water-diluted translation mixture, and 2 µL of 10× DNase I buffer for 30 min at 37 °C. DNase I was inactivated by heating at 75 °C for 10 min, following the addition of EDTA to 2.5 mM final concentration. The resulting reaction was diluted fivefold with water and 2 µL was combined with an equal volume of 1 µM reverse oligonucleotide targeting rRNA. The annealing reaction was performed at 78 °C for 8 min and allowed to slowly cool to RT. For cDNA synthesis 4 µL of annealing reaction was supplemented with 1 µL of 10× buffer for Avian Myeloblastosis Virus reverse transcriptase (AMV RT), 0.25 µL of dNTP mix (10 mM each), and 2 U of AMV RT (NEB, #M0277). The cDNA synthesis was performed at 50 °C for 45 min followed by 85 °C for 5 min for inactivation of reverse transcriptase. For qPCR, 1.25 µL or 2.5 µL of RT reaction was added into the well of 384-well plate (PerkinElmer, #6007290) containing 11.2 µL or 10 µL, respectively, of premix composed of 6.25 µL of Platinum™ SYBR™ Green qPCR SuperMix-UDG (Thermofisher), 0.25 µL of each oligonucleotide (10 µM stock concentration), 0.025 µL of ROX dye (ThermoFisher), 0.075 µL of DMSO, and 5 or 3.75 µL of water, respectively. The standard cycling program (95 °C, 12 min for initial denaturing, 40 cycles of 95 °C for 15 s, 60 °C for 1 min) was used followed by melting curve analysis using the default program of Applied Biosystems® ViiA™ 7 Real-Time PCR System.

Preparation of affinity-clamp-coupled resin (AC-resin)

The affinity-clamp protein (PDZ-fibronectin fusion protein, PDB: 3CH8) harboring C-terminal cysteine residue (Supplementary Table 6) was purified from BL21(DE3)RIL by Ni2+-affinity chromatography followed by gel filtration. Briefly, following induction with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at OD600 0.8, protein expression was carried out ON at 20 °C. Cells were disrupted using a continuous flow mode cell disruptor (Constant Systems) and protein was purified by Ni2+-affinity chromatography using standard buffers followed by gel-filtration chromatography on a Superdex 75 column (GE Healthcare) in 50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 1 mM EDTA, 2 mM TCEP. The protein sample was adjusted to 10 mg/mL in coupling buffer (50 mM Tris-HCl, 5 mM EDTA, 25 mM TCEP-KOH (pH 8.0), pH 8.5) and added to saturation to UltraLink® iodoacetyl resin (ThermoFisher, #53155) pre-washed with the coupling buffer. Following 1 h coupling reaction at room temperature, the resin was settled, washed with coupling buffer, blocked with 50 mM l-cysteine for 30 min. The AC-coated resin was washed again with 1 M NaCl and PBS and stored in PBS containing 0.1 mg/ml of BSA and 2 mM NaN3 as 50% (vol/vol) suspension at 4 °C. By titration of the resin amount derived from 50 µL of 50% (vol/vol) suspension with RGSIDTWV peptide, the binding capacity of the AC-resin was found to be 27 nanomoles of ligand per milliliter of settled resin gel or per 2 mL of 50% (vol/vol) suspension (Supplementary Fig. 1). For details, refer to a procedure overview in the Supplementary Methods.

Preparation of E. coli S30 extract and translation reaction assembly

For details, refer to a procedure overview in the Supplementary Methods. Briefly, E. coli S30 extract was prepared as described by Schwarz et al.68 with minor modifications. Briefly, cells were cultivated in 5–10 L of filter-sterilized TBGG media (tryptone 12 g/L, yeast extract 24 g/L, glycerol 8 mL/L, glucose 1 g/L, KH2PO4 2.31 g/L, K2HPO4 2.54 g/L) to OD 3.5, pre-chilled with 5 × 200 or 10 × 200 mL of –80 °C pre-frozen packs of LB broth and spun at 2.5 kg for 15 min. The cell pellet from the final wash was resuspended in 200 % (vol/wt) of S30B buffer and disrupted using fluidic disruption (Constant Systems, continuous flow mode) at 20 kpsi at 4 °C. Cell homogenate was cleared by two consecutive 30 min centrifugation steps at 30 kg at 4 °C while collecting the top ¾ of supernatant every time. The final supernatant was adjusted to 0.4 M with 5 M NaCl followed by 45 min incubation at 42 °C in a water bath. Following incubation, the cell homogenate was transferred to dialysis tubing (12–14 kDa cutoff, SpectrumTM Labs) and dialyzed for 2 h against 4 L of cold S30C buffer at 4 °C followed by ON dialysis against the same volume of fresh buffer. The dialyzed extract was centrifuged for 30 min at 30 kg at 4 °C and top ¾ of supernatant was collected, aliquoted, and frozen in LN2 for −80 °C storage. The translation reaction was assembled using 35% (vol/vol) S30 extract (in 10 mM Tris-acetate pH 8.2, 14 mM Mg(OAc)2, 0.6 mM KOAc, 0.5 mM DTT), 40% (vol/vol) of ×2.5 Feeding Solution (236 mM HEPES-KOH pH 7.4, 12.5 mM Mg(OAc)2, 375 mM KOAc, 5% PEG 8000, 12.5% glycerol, 5 mM NaN3, 0.015% Tween 20, 5 mM DTT, 2.5× protease inhibitor (cOmplete™ EDTA-free, Roche), 0.25 mg/mL folinic acid, 2 mM of each rNTP with an extra 1 mM of ATP, 38 mM of acetyl phosphate, 68 mM of creatine phosphate, 1.25 mM of each amino acid with extra 2.5 mM for Arg, Cys, Trp, Asp, Met, Glu), 0.05 mg/mL T7 RNA polymerase, 45 U/mL creatine phosphokinase, 0.05 mg/mL of TVMV protease and 20 nM of plasmid template. Translation of disulfide-rich proteins was performed in reactions supplemented with the extra 7.5 mM DTT and constituted with 17.5% (vol/vol) of S30 extract and 17.5% (vol/vol) of the extract buffer. Cyclosporin A (Sigma-Aldrich, #PHR1092) or Rapamycin (Sigma-Aldrich, #A3782) were supplemented to the respective translation reactions at 10 μM final concentration.

Preparation of Leishmania cell-extract and translation reaction assembly

A Leishmania-based transcription–translation system (LTE) was prepared as described by Kovtun et al.40. Briefly, Leishamnia tarentolae cells were expanded in 5-L conical flasks at 26.5 °C, 74 rpm agitation in a 1 L per flask of TBGG media (as for S30 extract, supplemented with hemin). Cells were harvested at 1.0–1.2 × 108 cells/ml, pelleted at 2.5 kg, and resuspended to 1010 cells/ml in a buffer containing 45 mM HEPES-KOH pH 7.6, 250 mM Sucrose,100 mM KOAc, 3 mM Mg(OAc)2 followed by disruption in a nitrogen cavitation device (70 bar N2, 45 min equilibration at 4 °C). Following two sequential 10,000 × g and 30,000 × g centrifugation, the top 2/3 of the final supernatant was collected and subjected to gel filtration on PD-10 Superdex 25 column (GE Healthcare) into fresh elution buffer (EB; 45 mM HEPES-KOH pH 7.6, 100 mM KOAc, 3 mM Mg(OAc)2). The 2.5 V of buffer-exchanged lysate was then supplemented with 1 V of 5x feeding solution containing 6 mM ATP, 0.68 mM GTP, 22.5 mM Mg(OAc)2, 1.25 mM spermidine, 10 mM DTT, 200 mM creatine phosphate, 100 mM HEPES-KOH pH 7.6, 5% (vol/vol) PEG 3000, 5.25× protease inhibitor cocktail (Complete™ EDTA-free, Roche), 0.68 mM of each amino acid, 2.5 mM rNTP mix (ATP, GTP, UTP and CTP), 0.05 mM anti-splice leader DNA oligonucleotide (αSL oligo, Supplementary Table 7), 0.5 mg/ml T7 RNA polymerase, 200 U/ml creatine phosphokinase, snap-frozen and stored at −80 °C. Transcription–translation reaction was assembled by adjusting 30 μl of supplemented lysate to 100 μl final reaction volume with  addition of plasmid template to final 20–40 nM, extra 5% of glycerol, 2 mM NaN3, and 0.005% Tween 20.

Translation in PURE and HeLa-based cell-free systems

Protein synthesis in a fully reconstituted PURE (Protein synthesis Using Recombinant Elements) translation system was performed using PURExpress In Vitro Protein Synthesis Kit (NEB, #E6800S) according to the manufacturer’s protocol. HeLa-based translation system was assembled using 1-Step Human Coupled IVT Kit (ThermoFisher, #88881) according to the manufacturer’s instructions. HeLa-based translation reactions were performed in 100 μL at 30 °C for 6 h.

Assembly of resin-assisted translation reactions

The resin-assisted translation was performed for Ec CFS, PURE, and LTE. For details regarding the assembly of AC-resin-assisted Ec CFS also refers to a procedure overview in Supplementary Methods. Briefly, the amount of AC-coated resin corresponding to 40 μl of 50% (vol/vol) resin suspension in PBS was used to supplement 100 μl of transcription–translation reaction either co- or post-translationally. Prior to reaction in PURE and Ec CFS, the resin was washed and equilibrated with the reaction buffer consisting of 35% (vol/vol) of the S30-extract buffer and 40% of the feeding solution. For LTE reaction the resin was equilibrated in a buffer consisting of 21% EB and 8.5% of LTE feeding solution. For post-translational product pulldown, AC-coated resin was supplemented in one reaction volume of a buffer containing 20 mM Tris-HCl pH 7.5, 1 M NaCl, 0.05% Tween 20 to terminate the translation reaction and allow product capture. In parallel, resin-assisted translation reaction was supplemented with 1×V of the same buffer and both mixtures were incubated for another 1 h at RT with agitation. Following incubation, the mixtures were transferred to filter-bottom tubes, flow-throughs were separated and the resins carrying immobilized peptide translation products were washed with six alternating sessions of water and Wash Buffer (50 mM Tris-HCl, 500 mM NaCl, 0.1% Tween 20, pH 7.5) while for immobilized protein products the water was replaced with Neutral Buffer (NB) (20 mM Tris-HCl, 20 mM NaCl, pH 7.5).

Strep-Tactin-resin-assisted translation reactions were prepared in the same way as described for AC-coated resin, with minor exceptions. Briefly, the amount corresponding to 20 µL of 50% (vol/vol) suspension of Commercial Strep-Tactin XT (IBA Lifesciences) resin was used to supplement 100 µL of translation reaction (ligand-binding capacity of Strep-Tactin-coated resin was found to be 50 nmol of ligand per ml of settled resin gel, corresponding to 2 ml of 50% (vol/vol) resin suspension in PBS). For post-translational product pulldown, following the completion of translation reaction, Strep-Tactin-coated resin was supplemented into translation in one reaction volume of a buffer containing 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.05% Tween 20. In parallel, resin-assisted translation reaction was supplemented with 1xV of the same buffer and both mixtures were incubated for another 1 h at RT with agitation. The same buffer was used as a Wash Buffer in combination with NB (20 mM Tris-HCl, 20 mM NaCl, pH 7.5) for six alternating washing sessions. Captured products were eluted from Strep-Tactin-coated resin with elution buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.25 mM EDTA) containing 50 mM biotin in two consecutive steps with 15 min incubation at 30 °C with 1400 rpm agitation at each step.

Peptide translation in resin-assisted CFSs

Peptide synthesis was performed at 27 °C for 3 h in LTE, and at 32 °C for 3 h in Ec CFS and 6 h in the PURE translation system. Affinity-clamp-assisted translation reactions were assembled and processed as described in the previous “Methods” section (for details also refer to a procedure overview in the Supplementary methods). RGS-tagged peptides were eluted from the AC-resin either with DMSO or 0.2% TFA, or a mixture of both at indicated proportions. The resin was incubated with 1 V of elution buffer relative to 50% suspension for 20 min with vigorous shaking at RT followed by centrifugation for 2 min at 17 kg. The elution step could be repeated three times followed by snap freezing and lyophilization of combined elution. To perform on-resin peptide reduction the resin was incubated with 50 mM DTT in 0.1 M NH4HCO3, pH 8.5 at RT for 30 min followed by washing and oxidative folding in a buffer containing 10 mM or less glutathione (GSH) in 0.1 M NH4HCO3 pH 8.5 at RT for 12 or 48 h with exception for kalata B1 (Supplementary note 6). For further analysis, peptides were eluted with 1 V of 0.2% TFA in three elution sessions followed by lyophilization. For more details, refer to a procedure overview in the Supplementary Methods.

Translation and on-resin refolding for proteins free of disulfide bonds

Translation was performed in Ec CFS and LTE in 100 μl reaction volume at 28 °C and 27 °C for 3 h, respectively. Affinity-clamp- and Strep-Tactin-assisted translation reactions were assembled and processed as described above under the “Assembly of resin-assisted translation reaction”. Proteins were eluted from Strep-Tactin-resin with an elution buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.25 mM EDTA) containing 50 mM biotin. Resin amount corresponding to 6 µL of 50% suspension (vol/vol) was eluted with 20 µL of buffer in two consecutive elution steps. For refolding of Strep-Tactin-resin-immobilized proteins the given amount of resin was incubated with 40 μl of refolding buffer (20 mM Tris-HCl, 2.5 mM DTT, 20 mM NaCl, 0,125% Tween 20, pH 7.5) containing either 0.8 or 1.6 M of guanidine hydrochloride (GndHCl) for 2 h at RT with 1400 rpm agitation in top-bench thermomixer (Eppendorf). Guanidine hydrochloride was originally prepared as 8 M stock based on neutral buffer. Treatment of Affinity-clamp-resin-immobilized proteins was performed in a buffer (20 mM Tris-HCl, 2.5 mM DTT, 1 M NaCl, 0,125% Tween 20, pH 7.5) containing 2 M GdnHCl for 6 h or 12 h at RT with 1400 rpm agitation. Control resins were incubated in the neutral buffer (NB) containing 20 mM Tris-HCl, 2.5 mM DTT, 20 mM NaCl, 0,125% Tween 20, pH 7.5. Following incubation, the refolding reactions were diluted twofold in two consecutive dilution steps with 1 h incubation following each dilution. Finally, the resins were extensively washed with the neutral buffer (20 mM Tris-HCl, 20 mM NaCl, 0.05% Tween 20, pH 7.5). For details, refer to a procedure overview in the Supplementary Methods.

AC-assisted translation, on-resin refolding, and pull-down analysis for disulfide-rich proteins

The antibody fragments and the CoV2-RBD (Supplementary Table 3) were translated in 20 μl of Ec CFS, 50 μl of LTE at 25 °C for 4 h and in 100 μl of HeLa-based translation system at 30 °C for 6 h. Ec CFS was modified with adjustment of DTT concentration to 10 mM and the use of S30 extract at twofold dilution. For details also refer to a procedure overview in the Supplementary Methods. Ec CFS and LTE translation reactions (performed in 20 μl and 50 μl, respectively) were supplemented with AC-coated resin amount corresponding to 8 μL and 20 μL of 50% (vol/vol) resin suspension co- and post-translationally while only post-translational pulldown was performed following translation in HeLa-based translation system. On-resin refolding for proteins immobilized co-translationally was performed by incubation of resin-bound protein fractions with a buffer containing 2 M guanidine hydrochloride (GdnHCl), 50 mM Tris-HCl, 1 M NaCl, 100 mM DTT, pH 7.5 for 2 h at RT followed by exchange into the buffer containing 1 M GdnHCl, 50 mM Tris-HCl, 0.5 M NaCl, and 10 mM reduced glutathione (GSH), pH 7.5, followed by 16 h incubation at RT with 1400 rpm agitation. Guanidine hydrochloride was originally prepared as 8 M stock based on neutral buffer. Control protein samples were incubated with a neutral buffer (NB) containing 20 mM Tris-HCl, 20 mM NaCl, pH 7.5. Following 16 h incubation, the mixtures were diluted by NB in two consequent twofold dilution steps and additionally incubated at RT for 1 h after each dilution step followed by the final resin washing step. Full denaturing/renaturing of antibody fragments was carried out in a buffer containing 6 M GdnHCl, 50 mM TrisHCl, pH 7.5, 1 M NaCl, and 100 mM DTT for 2 h at room temperature followed by overnight dialyzing against buffer lacking denaturant (50 mM Tris-HCl, 0.5 M NaCl, pH 7.5) at 4 °C. For details refer to a procedure overview in the Supplementary Methods. Pull-down assay was performed with 50 µL of 10 µM solution of respective antigens such as GFP (purified from pOPINE in-house), Human serum albumin (Sigma-Aldrich, #A3782), human IFNα-1b (GenScript, #Z02866), human IFNα-2a (Shenandoah Biotechnology, #100-54-100UG), human IL-6 (Shenandoah Biotechnology, #100-10-100UG) or SARS-COV2-RBD antibody (Sanyou Biopharmaceuticals, #AHA004). Incubation was conducted at RT for 3 h. After washing with neutral buffer, the beads were boiled at 95 °C for 5 min with 2×LDS sample buffer (Invitrogen™ 4X Bolt™, #B0008) and eluted fractions were analyzed on SDS-PAGE.

DHFR activity assay

Fluorescence DHFR activity assay was performed by monitoring the change in NADPH fluorescence (Ex 340 nm, Em 375 nm) upon its conversion to NADP+ in 200 µL of buffer containing 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM Mg(OAc)2, 0.012% Tween 20, 2.5 mM DTT in a black 96-well plate (Corning). Reactions were started simultaneously by pipetting the substrate mixture containing NADPH and dihydrofolic acid to 80 µM and 70 µM final concentration, respectively. Fluorescence changes were recorded for 4 h. Protein fractions bound to affinity-clamp-coated resin amount corresponding to 12.5 µL of 50% suspension (vol/vol) were directly used in the assay. Protein fractions from Strep-Tactin-assisted translation reactions were eluted from the resin corresponding to 6 µL of 50% suspension in two consecutive 20 µL elution steps (40 µL total elution volume). Pure recombinant E. coli DHFR was used to calibrate the assay (Supplementary Fig. 16B). Initial rates were derived from the fitting of the respective kinetic curves with polynomial or linear functions in Excel.

Colorimetric DHFR activity assay was used to monitor the activity of DHFR-calmodulin biosensor in response to a peptide inhibitor (Supplementary Note 8). The assay was performed by monitoring the decrease in absorbance of NADPH at 340 nm at 25 °C in 1 mL of 20 mM NaCl, 20 mM KH2PO4 pH 7.5 buffer containing 10 nM of chimeric DHFR-calmodulin sensor, 0.2 µM of M13 calmodulin-binding peptide, 80 µM NADPH, 67 µM dihydrofolic acid, 100 µM CaCl2 and 2 µM of either reduced or oxidized HT-1-RGS fusion. The changes in absorbance were recorded on Cary 50 UV–Vis spectrophotometer (Varian Inc.). Only oxidized in vitro translated HT-1 inhibited the biosensor confirming the installation of native disulfide bridges critical for HT-1 functionality (Supplementary Fig. 11C).

Peptide mass spectrometry

LC-MS/MS analysis was performed on Nexera UHPLC (Shimadzu, Japan) interfaced with a TripleTOF 5600 mass spectrometer (ABSCIEX, Canada) equipped with a duo electrospray ion source. Lyophilized peptide 100–200 ng was dissolved in 20 µL of 0.1% formic acid. 15 µL of peptide sample was injected onto a 2.1  × 100 mm Zorbax C18 1.8 µm column (Agilent) for analytical chromatography. Chromatography was performed in the eluent system of 0.1% formic acid in water (A) and 0.1% formic acid in 90% acetonitrile (B), at 0.2 mL/min flow rate using the following pump settings: 50 min 1–40% B, 8 min 40–98% B, 3 min 98% B, 3 min 98-1% B. The following settings were used: the ion spray voltage was set to 5500 V, de-clustering potential - to 100 V, curtain gas flow – to 25, nebulizer gas 1 - to 50, nebulizer gas 2 - to 60, interface heater - to 150 °C, and the turbo heater - to 500 °C. The mass spectrometer acquired 200 ms full-scan TOF-MS data followed by up to 10200 ms full-scan product-ion data acquisition in an Information Dependent Acquisition mode. Full-scan TOF-MS data were acquired over the mass range 400–7000 and for product-ion MS/MS 200–3500. Ions observed in the TOF-MS scan exceeding a threshold of 100 counts and a charge state of +2 to +5 were set to trigger the acquisition of product-ion MS/MS spectra of the resultant 10 most intense ions. The data were acquired and processed using Analyst TF 1.6 software (ABSCIEX, Canada).

MALDI-TOF

Peptide samples were initially analyzed using the 5800 MALDI-TOF/TOF Mass Spectrometer (Applied Biosystems). Peptides were made up in 70% acetonitrile with 0.1% formic acid in water and mixed 1:1 with α-cyano-4-hydroxycinnamic acid matrix (10 mg/mL) in the same solvent. Then 0.8 µL (10–100 ng) of each sample was spotted onto a stainless-steel target and allowed to air dry. Spectra were obtained in positive ion, linear mode using an accelerator voltage of 20 kV, grid voltage of 64%, and delay time of 350 ns. Each spectrum consisted of 200–500 shots from random target positions. Spectra were processed using Data Explorer software.

Western blot protocol

Western blot was performed as described previously69 with minor modifications. Briefly, protein samples were heated at 95 °C for 5 min with 2xSDS loading buffer (Invitrogen) and resolved on 12% SDS-PAGE (Invitrogen). Following electrophoresis, the proteins were transferred to PVDF membrane 0.2 µm (Millipore). Primary mouse anti-GFP antibodies (dilution WB 1:1000) (#11814460001, Roche) and secondary goat anti-mouse IgG (H + L) conjugated with HRP (dilution WB 1:5000) (Cat. No. G-21040, Life Technologies) were used for immunoblotting. Bound primary antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies and the Super Signal West Dura ECL detection reagent (Life Technologies). The specific protein bands were visualized using the Super Signal West Dura ECL detection reagent (Life Technologies) and imaged using the ChemiDoc Imaging System (Bio-rad, Gladesville, New South Wales, Australia).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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