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Materials and Methods\n\n4.1. Construction of the Expression Plasmid\nA bicistronic operon for the simultaneous expression of human Tα1 (UniProt P06454, residues 2–29) and the E. coli N-acetyltransferase RimJ (UniProt P0A948) flanked by the restriction sites NdeI and HindIII was prepared using gene synthesis (Thermofisher Scientific, Regensburg, Germany). To this end, the coding sequence of human Tα1 was codon-optimized for expression in E. coli and linked to the RimJ structural gene via the nucleotide sequence GCCTGAAGAGCAGAAAATAAA comprising a “GCC” alanine codon, the opal stop codon “TGA”, a SapI restriction site for insertion of the PAS gene cassette, and a ribosome-binding site (RBS). The entire gene fragment was subcloned via NdeI and HindIII on a derivative of pASK75 [36] for cytoplasmic expression under control of the tetracycline promoter/operator (tetp/o). Subsequently, a substantially non-repetitive sequence-verified PAS gene cassette [34] encoding a PAS#1 polypeptide of 601 amino acids was inserted via the SapI restriction site to create the expression vector for the C-terminally PASylated thymosin α1 (Tα1-PAS), pASK75-Tα1-PAS#1(600)/RimJ. To construct a plasmid for the bacterial production of an N-terminally PASylated Tα1 (PAS-Tα1), the synthetic gene of human Tα1 was amplified via PCR using the primers THY-For (5’-AGCTCTTCTGCCAGTGATGCAGCAGTTGATACC) and THY-Rev (5’-GCTCAAGCTTAGTTCTCGGCTTCTTCCAC). The PCR product was digested with SapI and HindIII and inserted via these restriction sites downstream of the PAS#1(600) sequence preceded by Met and Pro encoded on a suitable pASK75 plasmid derivative.\n\n4.2. Bacterial Production of Recombinant PASylated Tα1\nThe BL21 derivative E. coli strain NEBexpress® (New England Biolabs, Frankfurt am Main, Germany) was transformed with the plasmids pASK75-Tα1-PAS#1(600)/RimJ or pASK75-MP-PAS#1(600)-Tα1 from above. Two milliliters of Terrific Broth supplemented with 100 µg/mL ampicillin (TBamp) was inoculated with a single colony and grown overnight at 37 °C, 170 rpm. This overnight culture was used to inoculate a 50 mL preculture, which was grown under the same conditions until OD550 = 1.1 was reached. Subsequently, the 50 mL preculture was transferred into 2 L TBamp and incubated in a 5 L baffled flask at 37 °C and 100 rpm. After reaching OD550 = 0.6, the temperature was decreased to 26 °C and the cells were subsequently induced at OD550 = 1 with 200 µg/mL aTc for 15 h.\n\n4.3. Purification of PASylated Tα1\nBacteria were harvested by centrifugation and disrupted in the presence of 100 mM citric acid using an EmulsiFlex C5 cell homogenizer (Avestin, Mannheim, Germany). After centrifugation for 20 min (39,200× g) at 16 °C, the supernatant was filter-sterilized using a 0.45 µm syringe filter (Sartorius, Göttingen, Germany). The clear filtrate was adjusted with ammonium sulfate to 30% saturation, stirred for 30 min at RT, and centrifuged for 30 min (39,200× g) at RT. The precipitate was solubilized in subtractive AEX buffer (sAEX; 20 mM Tris/HCl, 1 mM EDTA, pH 8.5), filter-sterilized, and dialyzed twice against a 100-fold volume of sAEX buffer at 4 °C. Subsequent chromatography steps were performed on an ÄKTA Explorer 100 system (GE Healthcare, Freiburg, Germany) operated at a flow rate of 5 mL/min. The dialyzed sample (Tα1-PAS or PAS-Tα1) was first applied to a 5 mL TOYOPEARL NH2-750F column (Tosoh Bioscience, Griesheim, Germany) equilibrated with the sAEX buffer. The flow-through containing the PASylated Tα1 was collected and dialyzed twice against a 100-fold volume of the CEX buffer (20 mM Na-citrate, pH 3.0) at 4 °C. The protein solution was then applied to an 85 mL TOYOPEARL Sulfate-650F column (Tosoh Bioscience) equilibrated with the CEX buffer. PAS-Tα1 quantitatively bound to the column and the pure protein was eluted using a NaCl concentration gradient (0–250 mM over 2 column volumes). In the case of Tα1-PAS, the CEX flow-through fraction contained the N-terminally acetylated protein, whereas the non-acetylated Tα1-PAS stayed bound to the column and could again be eluted in a NaCl concentration gradient. The acetylated Tα1-PAS fraction from the flow-through was dialyzed twice against AEX buffer (20 mM ethanolamine/HCl, pH 10) and then applied to a 100 mL Fractogel® EMD TMAE (M)strong anion exchanger (Merck Millipore, Burlington, MA, USA) equilibrated with the AEX buffer. This time, highly pure acetylated Tα1-PAS was eluted using a NaCl concentration gradient (0–200 mM over 2 column volumes) in the AEX buffer. Both purified PASylated peptides were dialyzed 5 times against a 200-fold volume of water, lyophilized, and stored at −20 °C until further use.\n\n4.4. Analytical Size Exclusion Chromatography\nAnalytical SEC was performed on a 24 mL Superose® 6 10/300 GL column (GE Healthcare) using an ÄKTA Explorer 10 system operated at a flow rate of 0.5 mL/min with PBS as the running buffer. To determine the apparent molecular size, purified PAS-Tα1 or PAS-Tα1 (100 µL) was applied to the column and the elution volume was used for linear interpolation from a half-logarithmic calibration line obtained using the reference proteins thyroglobulin (octamer), thyroglobulin (tetramer), apoferritin, β-amylase, alcohol dehydrogenase (tetramer), transferrin, ovalbumin, and carboanhydrase as well as blue dextran (all from Merck, Darmstadt, Germany).\n\n4.5. Endotoxin Quantification\nTα1-PAS samples were diluted to 1 mg/mL in endotoxin-free water (Veolia Water Technologies, Celle, Germany), heated to 90 °C for 5 min, and, after cooling down to RT, bacterial endotoxins were quantified using an Endosafe® system (Charles River Laboratories, Wilmington, MA, USA) with an FDA-licensed PTS™ cartridge (10–0.1 EU/mL sensitivity).\n\n4.6. Western Blot Analysis\nThe whole cell protein extract was applied to a 4–12% SurePAGE™ Bis–Tris gradient gel (Genscript, Piscataway, NJ, USA) with 3-(N-morpholino)propanesulfonic acid (MOPS) as running buffer and electrotransferred onto a nitrocellulose membrane using an iBlot 1 dry blotting system (ThermoFisher, Waltham, MA, USA). After washing 3 times with PBS supplemented with 0.1% v/v Tween 20 (PBS/T), the membrane was incubated with 1 µg/mL anti-PAS antibody in PBS/T for 1 h at RT. The membrane was washed 3 times with PBS/T and incubated with a 1:5000 dilution of a goat anti-mouse IgG (Fc-specific) alkaline phosphatase (AP) conjugate (Merck) for 1 h. After washing twice with PBS/T and twice with PBS, the blot was developed by a chromogenic reaction using BCIP (37.5 µg/mL) and NBT (150 µg/mL) in alkaline phosphatase buffer (100 mM Tris/HCl, pH 8.8, 100 mM NaCl, 5 mM MgCl2).\n\n4.7. Reverse-Phase Chromatography (RPC) and ESI Mass Spectrometry\nDirectly after the CEX chromatography, a 200 µl sample of PAS-Tα1 or PAS-Tα1 was adjusted to 2% v/v acetonitrile, 0.1% v/v formic acid and applied to a Resource RPC 1 mL column (GE Healthcare) equilibrated with 2% v/v acetonitrile, 0.1% v/v formic acid. The protein was eluted using a concentration gradient of up to 80% v/v acetonitrile, 0.1% v/v formic acid over 20 column volumes at a flow rate of 2 mL/min while spectrophotometrically monitoring protein elution at 225 nm. The eluted protein fraction was directly injected into a maXis quadrupole time of flight (Q-TOF) instrument (Bruker Daltonics, Bremen, Germany) operated in the positive ion mode. Raw data was deconvoluted using the MaxEntX algorithm (Bruker Daltonics).\n\n4.8. Pharmacokinetic Analysis in Rats\nA PK study in female Wistar rats at 8–9 weeks of age was conducted at the Aurigon Toxicological Research Center (ATRC, Dunakeszi, Hungary). Up to 3 animals per cage were housed in a controlled environment at 22 ± 3 °C with a relative humidity of 50 ± 20%, 12 h light and 12 h dark. Endotoxin-free purified Tα1-PAS (3.4 mg/kg) was administered subcutaneously via a single injection into the rat dorsal area. Blood samples (100 µL) were taken from 5 animals each at various time points. Following collection in tubes containing tri-potassium ethylenediaminetetraacetic acid (K3-EDTA) (Greiner Bio-One, Frickenhausen, Germany), samples were centrifuged at room temperature for 10 min (3000× g) and the resulting plasma was stored at −15 to −30 °C. Tα1-PAS in these samples was quantified using sandwich ELISA (see below) and the data were analyzed using the Phoenix WinNonlin 6.3 software (Certara, Princeton, NJ, USA) using a one-compartment model assuming 1st order absorption and elimination.\n\n4.9. Quantification of Purified Tα1-PAS\nDue to the absence of aromatic side chains, PASylated Tα1 does not absorb light at 280 nm. Thus, for reliable quantification via the peptide backbone absorption, an extinction coefficient at 225 nm was determined. To this end, highly pure Tα1-PAS was lyophilized from water, weighed, and dissolved in a defined volume of water. The concentration of this Tα1-PAS solution was additionally quantified by measuring the backbone peptide groups using UV absorption at 205 nm using a generic calculated extinction coefficient [75], which led to a fair agreement (±6%). The absorbance at 225 nm was measured for various dilutions of the gravimetrically prepared peptide stock solution and plotted against the concentration. The linear range was fitted by a straight line, resulting in an extinction coefficient of 253,057 M−1⋅cm−1. While most buffers strongly absorb light at 205 nm, such a spectral overlap is usually absent at 225 nm.\n\n4.10. ELISA Quantification of PASylated Tα1\nA 96-well Nunc Maxisorb ELISA plate (ThermoFisher) was coated with 100 µg/mL anti-PAS antibody in PBS at 4 °C overnight. After washing twice with PBS/T, free binding sites were blocked with 3% w/v bovine serum albumin (BSA) in PBS/T at room temperature for 1 h. After washing 3 times with PBS/T, the rat plasma samples were applied in dilution series in PBS/T supplemented with 0.5% (v/v) plasma from an untreated animal (to maintain a constant proportion of rat plasma constituents). In the same manner, a standard curve was prepared using dilution series of purified Tα1-PAS (used for spiking rat plasma) at defined concentrations. After incubation for 1 h at RT, wells were washed 3 times with PBS/T. To detect bound Tα1-PAS, wells were incubated for 1 h with 50 µl of a 1 µg/mL PBS/T solution of the second anti-PAS antibody conjugated with alkaline phosphatase using the Lightning-Link alkaline phosphatase antibody labeling kit (BioTechne, Wiesbaden, Germany). After washing twice with PBS/T and twice with PBS, the enzymatic activity was detected using p-nitrophenyl phosphate (0.5 mg/mL). To this end, the plate was incubated for 20 min at 30 °C, the absorbance was measured at 405 nm using a SpectraMax M5e microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA) and the Tα1-PAS concentrations in rat plasma samples were quantified by comparison with the standard curve."}

LitCovid-sentences

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7},"obj":"Sentence"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/ontology/tao.owl#"}],"text":"4. Materials and Methods\n\n4.1. Construction of the Expression Plasmid\nA bicistronic operon for the simultaneous expression of human Tα1 (UniProt P06454, residues 2–29) and the E. coli N-acetyltransferase RimJ (UniProt P0A948) flanked by the restriction sites NdeI and HindIII was prepared using gene synthesis (Thermofisher Scientific, Regensburg, Germany). To this end, the coding sequence of human Tα1 was codon-optimized for expression in E. coli and linked to the RimJ structural gene via the nucleotide sequence GCCTGAAGAGCAGAAAATAAA comprising a “GCC” alanine codon, the opal stop codon “TGA”, a SapI restriction site for insertion of the PAS gene cassette, and a ribosome-binding site (RBS). The entire gene fragment was subcloned via NdeI and HindIII on a derivative of pASK75 [36] for cytoplasmic expression under control of the tetracycline promoter/operator (tetp/o). Subsequently, a substantially non-repetitive sequence-verified PAS gene cassette [34] encoding a PAS#1 polypeptide of 601 amino acids was inserted via the SapI restriction site to create the expression vector for the C-terminally PASylated thymosin α1 (Tα1-PAS), pASK75-Tα1-PAS#1(600)/RimJ. To construct a plasmid for the bacterial production of an N-terminally PASylated Tα1 (PAS-Tα1), the synthetic gene of human Tα1 was amplified via PCR using the primers THY-For (5’-AGCTCTTCTGCCAGTGATGCAGCAGTTGATACC) and THY-Rev (5’-GCTCAAGCTTAGTTCTCGGCTTCTTCCAC). The PCR product was digested with SapI and HindIII and inserted via these restriction sites downstream of the PAS#1(600) sequence preceded by Met and Pro encoded on a suitable pASK75 plasmid derivative.\n\n4.2. Bacterial Production of Recombinant PASylated Tα1\nThe BL21 derivative E. coli strain NEBexpress® (New England Biolabs, Frankfurt am Main, Germany) was transformed with the plasmids pASK75-Tα1-PAS#1(600)/RimJ or pASK75-MP-PAS#1(600)-Tα1 from above. Two milliliters of Terrific Broth supplemented with 100 µg/mL ampicillin (TBamp) was inoculated with a single colony and grown overnight at 37 °C, 170 rpm. This overnight culture was used to inoculate a 50 mL preculture, which was grown under the same conditions until OD550 = 1.1 was reached. Subsequently, the 50 mL preculture was transferred into 2 L TBamp and incubated in a 5 L baffled flask at 37 °C and 100 rpm. After reaching OD550 = 0.6, the temperature was decreased to 26 °C and the cells were subsequently induced at OD550 = 1 with 200 µg/mL aTc for 15 h.\n\n4.3. Purification of PASylated Tα1\nBacteria were harvested by centrifugation and disrupted in the presence of 100 mM citric acid using an EmulsiFlex C5 cell homogenizer (Avestin, Mannheim, Germany). After centrifugation for 20 min (39,200× g) at 16 °C, the supernatant was filter-sterilized using a 0.45 µm syringe filter (Sartorius, Göttingen, Germany). The clear filtrate was adjusted with ammonium sulfate to 30% saturation, stirred for 30 min at RT, and centrifuged for 30 min (39,200× g) at RT. The precipitate was solubilized in subtractive AEX buffer (sAEX; 20 mM Tris/HCl, 1 mM EDTA, pH 8.5), filter-sterilized, and dialyzed twice against a 100-fold volume of sAEX buffer at 4 °C. Subsequent chromatography steps were performed on an ÄKTA Explorer 100 system (GE Healthcare, Freiburg, Germany) operated at a flow rate of 5 mL/min. The dialyzed sample (Tα1-PAS or PAS-Tα1) was first applied to a 5 mL TOYOPEARL NH2-750F column (Tosoh Bioscience, Griesheim, Germany) equilibrated with the sAEX buffer. The flow-through containing the PASylated Tα1 was collected and dialyzed twice against a 100-fold volume of the CEX buffer (20 mM Na-citrate, pH 3.0) at 4 °C. The protein solution was then applied to an 85 mL TOYOPEARL Sulfate-650F column (Tosoh Bioscience) equilibrated with the CEX buffer. PAS-Tα1 quantitatively bound to the column and the pure protein was eluted using a NaCl concentration gradient (0–250 mM over 2 column volumes). In the case of Tα1-PAS, the CEX flow-through fraction contained the N-terminally acetylated protein, whereas the non-acetylated Tα1-PAS stayed bound to the column and could again be eluted in a NaCl concentration gradient. The acetylated Tα1-PAS fraction from the flow-through was dialyzed twice against AEX buffer (20 mM ethanolamine/HCl, pH 10) and then applied to a 100 mL Fractogel® EMD TMAE (M)strong anion exchanger (Merck Millipore, Burlington, MA, USA) equilibrated with the AEX buffer. This time, highly pure acetylated Tα1-PAS was eluted using a NaCl concentration gradient (0–200 mM over 2 column volumes) in the AEX buffer. Both purified PASylated peptides were dialyzed 5 times against a 200-fold volume of water, lyophilized, and stored at −20 °C until further use.\n\n4.4. Analytical Size Exclusion Chromatography\nAnalytical SEC was performed on a 24 mL Superose® 6 10/300 GL column (GE Healthcare) using an ÄKTA Explorer 10 system operated at a flow rate of 0.5 mL/min with PBS as the running buffer. To determine the apparent molecular size, purified PAS-Tα1 or PAS-Tα1 (100 µL) was applied to the column and the elution volume was used for linear interpolation from a half-logarithmic calibration line obtained using the reference proteins thyroglobulin (octamer), thyroglobulin (tetramer), apoferritin, β-amylase, alcohol dehydrogenase (tetramer), transferrin, ovalbumin, and carboanhydrase as well as blue dextran (all from Merck, Darmstadt, Germany).\n\n4.5. Endotoxin Quantification\nTα1-PAS samples were diluted to 1 mg/mL in endotoxin-free water (Veolia Water Technologies, Celle, Germany), heated to 90 °C for 5 min, and, after cooling down to RT, bacterial endotoxins were quantified using an Endosafe® system (Charles River Laboratories, Wilmington, MA, USA) with an FDA-licensed PTS™ cartridge (10–0.1 EU/mL sensitivity).\n\n4.6. Western Blot Analysis\nThe whole cell protein extract was applied to a 4–12% SurePAGE™ Bis–Tris gradient gel (Genscript, Piscataway, NJ, USA) with 3-(N-morpholino)propanesulfonic acid (MOPS) as running buffer and electrotransferred onto a nitrocellulose membrane using an iBlot 1 dry blotting system (ThermoFisher, Waltham, MA, USA). After washing 3 times with PBS supplemented with 0.1% v/v Tween 20 (PBS/T), the membrane was incubated with 1 µg/mL anti-PAS antibody in PBS/T for 1 h at RT. The membrane was washed 3 times with PBS/T and incubated with a 1:5000 dilution of a goat anti-mouse IgG (Fc-specific) alkaline phosphatase (AP) conjugate (Merck) for 1 h. After washing twice with PBS/T and twice with PBS, the blot was developed by a chromogenic reaction using BCIP (37.5 µg/mL) and NBT (150 µg/mL) in alkaline phosphatase buffer (100 mM Tris/HCl, pH 8.8, 100 mM NaCl, 5 mM MgCl2).\n\n4.7. Reverse-Phase Chromatography (RPC) and ESI Mass Spectrometry\nDirectly after the CEX chromatography, a 200 µl sample of PAS-Tα1 or PAS-Tα1 was adjusted to 2% v/v acetonitrile, 0.1% v/v formic acid and applied to a Resource RPC 1 mL column (GE Healthcare) equilibrated with 2% v/v acetonitrile, 0.1% v/v formic acid. The protein was eluted using a concentration gradient of up to 80% v/v acetonitrile, 0.1% v/v formic acid over 20 column volumes at a flow rate of 2 mL/min while spectrophotometrically monitoring protein elution at 225 nm. The eluted protein fraction was directly injected into a maXis quadrupole time of flight (Q-TOF) instrument (Bruker Daltonics, Bremen, Germany) operated in the positive ion mode. Raw data was deconvoluted using the MaxEntX algorithm (Bruker Daltonics).\n\n4.8. Pharmacokinetic Analysis in Rats\nA PK study in female Wistar rats at 8–9 weeks of age was conducted at the Aurigon Toxicological Research Center (ATRC, Dunakeszi, Hungary). Up to 3 animals per cage were housed in a controlled environment at 22 ± 3 °C with a relative humidity of 50 ± 20%, 12 h light and 12 h dark. Endotoxin-free purified Tα1-PAS (3.4 mg/kg) was administered subcutaneously via a single injection into the rat dorsal area. Blood samples (100 µL) were taken from 5 animals each at various time points. Following collection in tubes containing tri-potassium ethylenediaminetetraacetic acid (K3-EDTA) (Greiner Bio-One, Frickenhausen, Germany), samples were centrifuged at room temperature for 10 min (3000× g) and the resulting plasma was stored at −15 to −30 °C. Tα1-PAS in these samples was quantified using sandwich ELISA (see below) and the data were analyzed using the Phoenix WinNonlin 6.3 software (Certara, Princeton, NJ, USA) using a one-compartment model assuming 1st order absorption and elimination.\n\n4.9. Quantification of Purified Tα1-PAS\nDue to the absence of aromatic side chains, PASylated Tα1 does not absorb light at 280 nm. Thus, for reliable quantification via the peptide backbone absorption, an extinction coefficient at 225 nm was determined. To this end, highly pure Tα1-PAS was lyophilized from water, weighed, and dissolved in a defined volume of water. The concentration of this Tα1-PAS solution was additionally quantified by measuring the backbone peptide groups using UV absorption at 205 nm using a generic calculated extinction coefficient [75], which led to a fair agreement (±6%). The absorbance at 225 nm was measured for various dilutions of the gravimetrically prepared peptide stock solution and plotted against the concentration. The linear range was fitted by a straight line, resulting in an extinction coefficient of 253,057 M−1⋅cm−1. While most buffers strongly absorb light at 205 nm, such a spectral overlap is usually absent at 225 nm.\n\n4.10. ELISA Quantification of PASylated Tα1\nA 96-well Nunc Maxisorb ELISA plate (ThermoFisher) was coated with 100 µg/mL anti-PAS antibody in PBS at 4 °C overnight. After washing twice with PBS/T, free binding sites were blocked with 3% w/v bovine serum albumin (BSA) in PBS/T at room temperature for 1 h. After washing 3 times with PBS/T, the rat plasma samples were applied in dilution series in PBS/T supplemented with 0.5% (v/v) plasma from an untreated animal (to maintain a constant proportion of rat plasma constituents). In the same manner, a standard curve was prepared using dilution series of purified Tα1-PAS (used for spiking rat plasma) at defined concentrations. After incubation for 1 h at RT, wells were washed 3 times with PBS/T. To detect bound Tα1-PAS, wells were incubated for 1 h with 50 µl of a 1 µg/mL PBS/T solution of the second anti-PAS antibody conjugated with alkaline phosphatase using the Lightning-Link alkaline phosphatase antibody labeling kit (BioTechne, Wiesbaden, Germany). After washing twice with PBS/T and twice with PBS, the enzymatic activity was detected using p-nitrophenyl phosphate (0.5 mg/mL). To this end, the plate was incubated for 20 min at 30 °C, the absorbance was measured at 405 nm using a SpectraMax M5e microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA) and the Tα1-PAS concentrations in rat plasma samples were quantified by comparison with the standard curve."}