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Expression and Purification of Active Recombinant T7 RNA Polymerase from E. coli Donald C. Rio Cold Spring Harb Protoc; doi: 10.1101/pdb.prot078527 Email Alerting Service Subject Categories
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© 2013 Cold Spring Harbor Laboratory Press
Protocol
Expression and Purification of Active Recombinant T7 RNA Polymerase from E. coli Donald C. Rio
For large-scale transcription reactions or for cost savings, a laboratory may want to prepare its own recombinant T7-, SP6-, or T3-phage RNA polymerases. It is convenient to perform this preparation every 2–3 years and have a consistent and reliable source of phage RNA polymerase for many in vitro transcription reactions. In the protocol presented here, the recombinant plasmid expressing T7 RNA polymerase (RNAP) as a his6-tagged molecule is under an isopropyl β-D-1-thio-galactopyranoside (IPTG)-inducible promoter. The bacteria are lysed by sonication, the his6-tagged protein in the bacterial lysate is purified by binding to Ni-NTA agarose, and the resin is then extensively washed and eluted with imidazole. The purified enzyme is dialyzed against a glycerol-containing storage buffer and can then be stored for months or years at −20˚C.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Agar plates with LB medium and (50 µg/mL) ampicillin Ampicillin (sodium salt) (50 mg/mL) To 0.5 g ampicillin sodium salt (Sigma-Aldrich, A9518), add ddH2O to 10 mL. Filter through a 0.2-μm filter, and store at −20˚C.
Bacterial expression strain: BL21(DE3) competent cells CaCl2 (0.1 M) Filter through a 0.2-μm filter.
Glycerol (25%, v/v) Autoclave to sterilize.
Isopropyl β-D-1-thiogalactopyranoside (IPTG, 1.0 M) Leupeptin (5 mg/mL) Dissolve leupeptin in 100% DMSO and store in a polypropylene tube at −20˚C.
Lysozyme (10 mg/mL) Prepare fresh for each purification by mixing 100 mg of chicken egg white lysozyme with ddH2O to 10 mL.
Adapted from RNA: A Laboratory Manual, by Donald C. Rio, Manuel Ares Jr, Gregory J. Hannon, and Timothy W. Nilsen. CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2013 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078527
1094
Preparation of Active T7 RNA Polymerase
M9 salts (10×) Mix 68 g of Na2HPO4, 30 g of KH2PO4, and 10 g of NH4Cl, and adjust the volume to 900 mL with ddH2O. Filter through a 0.2-μm filter; aliquot in sterile bottles.
MgSO4 (1 M) Autoclave to sterilize.
NaCl Ni-NTA agarose (Qiagen 1000630) Ni-NTA elution buffer Ni-NTA lysis buffer Ni-NTA wash buffer Phenylmethysulfonyl fluoride (PMSF, 20 mg/mL) Dissolve PMSF in isopropanol and store in a dark glass bottle at −20˚C.
Sodium deoxycholate (8%, w/v) Prepare fresh for each purification by dissolving 0.8 g of deoxycholic acid, sodium salt in ddH2O.
T7 RNA polymerase expression plasmid: pT7-911Q (obtained from Thomas E. Shrader, Albert Einstein College of Medicine; Ichetovkin et al. 1997) pT7-911Q is the pQE-8 plasmid (Qiagen) with lacIQ in the XbaI site and His6T7-RNAP in the BamHI site. It has a PLac promoter/operator and confers ampicillin resistance (AmpR).
T7-RNAP storage buffer Tryptone Equipment
Autoclave Centrifuge bottles (250-mL or 500-mL) Centrifuges (Sorvall RC-3B with SS-34 rotor; Sorvall RC-5B with GS3 or SLA-1500 rotor; or equivalents) Centrifuge tubes (50-mL; Oak Ridge style) Chromatography column, glass (2.5-cm diameter × 10-cm length; Bio-Rad 737-4251) Cold room (4˚C) Conical tubes (25 mL, 50 mL) Culture tubes Dialysis equipment Equipment and 7.5% gel for SDS–PAGE Fernbach flasks (2 L) Freezer (−20˚C) Ice Incubator (37˚C) Rocker Screw-cap tubes (1.5 mL) Shaker (37˚C), for tubes and flasks Sonicator Spectrophotometer and cuvettes Toothpicks or wire loop, sterilized METHOD Bacterial Protein Expression This expression and purification procedure lasts 4 d, beginning with transformation and overnight incubation of competent bacterial cells. The transformed cells are used to begin starter cultures early on the second day, and
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078527
1095
D.C. Rio those cultures are later used to inoculate larger overnight cultures for protein expression. Lysis of the cells and protein purification takes place on day three, followed by a 24-h dialysis of the purified T7-RNAP.
1. Transform pT7-911Q into BL21-(DE3) competent cells. Plate on agar plates with LB medium and 50 µg/mL ampicillin. Incubate the plates overnight at 37˚C.
2. Prepare 900 mL of tryptone broth for each liter of culture by combining 10 g of tryptone, 5.5 g of NaCl, and 900 mL of ddH2O in a 2-L Fernbach flask. Autoclave for 20 min on a slow exhaust, liquid cycle. 3. Early the next day, prepare M9TB expression medium by adding the following to each flask of tryptone broth: 10× M9 salts 0.1 M CaCl2 1 M MgSO4 25% (v/v) glycerol 50 mg/mL ampicillin
100 mL 1 mL 1 mL 8 mL 0.8 mL
4. Start an inoculum by using a sterilized toothpick or wire loop to resuspend 2–3 fresh bacterial colonies (1 mm in diameter) in 1.0 mL of M9TB. Incubate the inoculum at 37˚C with shaking. It is important to use freshly transformed bacterial colonies obtained immediately after a transformation into BL21(DE3) competent cells. Do not store the plates at 4˚C.
5. At the end of the day, inoculate the Fernbach flasks. For each liter of M9ZB, use 100 µL of inoculum. Incubate the flasks overnight at 37˚C with shaking. 6. Check the OD600 of the cells in the flasks.
7. When the OD600 is 0.5–0.6, induce the cells in the flasks by adding 0.5 mL of 1 M IPTG per 1-L culture. 8. Continue to incubate the cells for 3 h at 37˚C with shaking.
9. Harvest the cells: in a Sorvall RC-5B centrifuge or equivalent, centrifuge the culture for 10 min in 500-mL bottles at 4000 rpm in a GS3 rotor or in 250-mL bottles at 7000 rpm in an SLA1500 rotor. Bacterial Lysis and Nickel-Agarose Affinity Purification
10. Resuspend the cells in 25 mL of lysis buffer per pellet. Transfer the cells to 50-mL conical tubes. 11. Add 1.0 mL of 10 mg/mL lysozyme, 50 µL of 20 mg/mL PMSF, and 20 µL of 5 mg/mL leupeptin. 12. Incubate the cells with rocking for 20 min at 4˚C.
13. Add 0.25 mL of 8% (w/v) sodium deoxycholate and incubate with rocking for 20 min at 4˚C.
14. Sonicate each pellet four times for 15 sec until dispersed. Keep the samples in ice during sonication. 15. Transfer the samples to 50-mL Oak Ridge centrifuge tubes. Centrifuge in a Sorvall SS-34 rotor at 15,500 rpm for 30 min at 4˚C. 16. Decant the supernatant into a clean bottle or flask, and keep it on ice.
17. Make a column of Ni-NTA agarose, using 10 mL of 50% slurry for each liter of starting cell culture. For example, for 2 L of cell culture, use 20 mL of 50% slurry.
18. Equilibrate the resin with ≥10 column volumes of lysis buffer. 19. Load the protein sample by gravity flow onto the column, and collect the flowthrough. 20. Wash the resin with three column volumes of lysis buffer followed by 10 column volumes of wash buffer. Collect the flowthrough. To remove all bacterial nuclease contamination from the purified protein, it is critical that you wash the column extensively with ≥10 column volumes of wash buffer before the imidazole elution.
1096
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078527
Preparation of Active T7 RNA Polymerase
21. Elute the T7-RNAP with 10 column volumes of elution buffer. Collect the eluent in aliquots of one column volume. 22. Check 5 µL of each fraction by SDS–PAGE on a 7.5% SDS-polyacrylamide gel. 23. Pool the peak fractions containing T7-RNAP (the protein runs at 100 kDa). 24. Dialyze the peak fractions against T7-RNAP storage buffer for 24 h at 4˚C, with three changes of 100 volumes each. 25. Quantitate the purified T7-RNAP by measuring the absorbance at 280 nm (A280). The estimated mg/mL = A280 × (dilution factor/1.3). Aliquot the enzyme in 100- to 200-μL aliquots into 1.5mL screw-cap tubes on ice in a cold room. 26. Store the purified T7-RNAP at −20˚C.
27. Determine the activity of the purified T7-RNAP. A practical way to determine activity is to perform in vitro transcription reactions with serially diluted enzyme from your preparation versus several amounts of commercially available enzyme; assay the incorporation of [α-32P]UTP by TCA precipitation or DEAE filter retention.
RELATED INFORMATION
The protein expression system described here is derived from the T7 promoter-driven system originally developed by Studier et al. (1990).
RECIPES Ni-NTA Elution Buffer
Reagent Ni-NTA lysis buffer (5×) β-Mercaptoethanol (14.1 M) Imidazole (1 M, pH 8.0) ddH2O
Quantity (for 200 mL)
Final concentration
40 mL 71 µL, add fresh 100 mL to 200 mL
1× 5 mM 500 mM
Ni-NTA Lysis Buffer
Reagent β-Mercaptoethanol (14.1 M) Imidazole (1 M, pH 8.0) ddH2O
Quantity (for 200 mL)
Final concentration
71 µL, add fresh 200 µL to 200 mL
5 mM 1 mM (pH 8.0)
Ni-NTA Lysis Buffer (5×)
Reagent Tris-HCl (1 M, pH 8.0) NaCl (5 M) Glycerol (100%)
Quantity (for 200 mL)
Final concentration (5×)
50 mL 20 mL 50 mL
250 mM 500 mM 25% (v/v)
Autoclave. Store for up to 6 mo at 4˚C. Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078527
1097
D.C. Rio
Ni-NTA Wash Buffer
Reagent Ni-NTA lysis buffer (5×) β-Mercaptoethanol (14.1 M) Imidazole (1 M, pH 8.0) ddH2O
Quantity (for 500 mL)
Final concentration
100 mL 177.5 µL, add fresh 5 mL to 500 mL
1× 5 mM 10 mM
T7-RNAP Storage Buffer
Reagent Potassium phosphate (0.2 M, pH 7.5) NaCl (5 M) Glycerol (100%) Dithiothreitol (DTT, 0.5 M) EDTA (0.25 M, pH 8.0) NaN3 powder (Sigma-Aldrich) ddH2O
Quantity (for 1 L)
Final concentration
100 mL 20 mL 500 mL 20 mL 0.4 mL 2g to 1 L
20 mM 100 mM 50% (v/v) 10 mM 0.1 mM 0.2% (w/v)
REFERENCES Ichetovkin IE, Abramochkin G, Shrader TE. 1997. Substrate recognition by the leucyl/phenylalanyl-tRNA-protein transferase. Conservation within the enzyme family and localization to the trypsin-resistant domain. J Biol Chem 272: 33009–33014.
1098
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185: 60–89.
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078527
Expression and Purification of Active Recombinant T7 RNA Polymerase from E. coli Donald C. Rio Cold Spring Harb Protoc; doi: 10.1101/pdb.prot078527 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Affinity and Immunoaffinity Chromatography (43 articles) Expression of Cloned Genes (67 articles) Molecular Biology, general (1002 articles) RNA (188 articles) RNA, general (165 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2013 Cold Spring Harbor Laboratory Press
Protocol
Expression and Purification of Active Recombinant T7 RNA Polymerase from E. coli Donald C. Rio
For large-scale transcription reactions or for cost savings, a laboratory may want to prepare its own recombinant T7-, SP6-, or T3-phage RNA polymerases. It is convenient to perform this preparation every 2–3 years and have a consistent and reliable source of phage RNA polymerase for many in vitro transcription reactions. In the protocol presented here, the recombinant plasmid expressing T7 RNA polymerase (RNAP) as a his6-tagged molecule is under an isopropyl β-D-1-thio-galactopyranoside (IPTG)-inducible promoter. The bacteria are lysed by sonication, the his6-tagged protein in the bacterial lysate is purified by binding to Ni-NTA agarose, and the resin is then extensively washed and eluted with imidazole. The purified enzyme is dialyzed against a glycerol-containing storage buffer and can then be stored for months or years at −20˚C.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Agar plates with LB medium and (50 µg/mL) ampicillin Ampicillin (sodium salt) (50 mg/mL) To 0.5 g ampicillin sodium salt (Sigma-Aldrich, A9518), add ddH2O to 10 mL. Filter through a 0.2-μm filter, and store at −20˚C.
Bacterial expression strain: BL21(DE3) competent cells CaCl2 (0.1 M) Filter through a 0.2-μm filter.
Glycerol (25%, v/v) Autoclave to sterilize.
Isopropyl β-D-1-thiogalactopyranoside (IPTG, 1.0 M) Leupeptin (5 mg/mL) Dissolve leupeptin in 100% DMSO and store in a polypropylene tube at −20˚C.
Lysozyme (10 mg/mL) Prepare fresh for each purification by mixing 100 mg of chicken egg white lysozyme with ddH2O to 10 mL.
Adapted from RNA: A Laboratory Manual, by Donald C. Rio, Manuel Ares Jr, Gregory J. Hannon, and Timothy W. Nilsen. CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2013 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078527
1094
Preparation of Active T7 RNA Polymerase
M9 salts (10×) Mix 68 g of Na2HPO4, 30 g of KH2PO4, and 10 g of NH4Cl, and adjust the volume to 900 mL with ddH2O. Filter through a 0.2-μm filter; aliquot in sterile bottles.
MgSO4 (1 M) Autoclave to sterilize.
NaCl Ni-NTA agarose (Qiagen 1000630) Ni-NTA elution buffer Ni-NTA lysis buffer Ni-NTA wash buffer Phenylmethysulfonyl fluoride (PMSF, 20 mg/mL) Dissolve PMSF in isopropanol and store in a dark glass bottle at −20˚C.
Sodium deoxycholate (8%, w/v) Prepare fresh for each purification by dissolving 0.8 g of deoxycholic acid, sodium salt in ddH2O.
T7 RNA polymerase expression plasmid: pT7-911Q (obtained from Thomas E. Shrader, Albert Einstein College of Medicine; Ichetovkin et al. 1997) pT7-911Q is the pQE-8 plasmid (Qiagen) with lacIQ in the XbaI site and His6T7-RNAP in the BamHI site. It has a PLac promoter/operator and confers ampicillin resistance (AmpR).
T7-RNAP storage buffer Tryptone Equipment
Autoclave Centrifuge bottles (250-mL or 500-mL) Centrifuges (Sorvall RC-3B with SS-34 rotor; Sorvall RC-5B with GS3 or SLA-1500 rotor; or equivalents) Centrifuge tubes (50-mL; Oak Ridge style) Chromatography column, glass (2.5-cm diameter × 10-cm length; Bio-Rad 737-4251) Cold room (4˚C) Conical tubes (25 mL, 50 mL) Culture tubes Dialysis equipment Equipment and 7.5% gel for SDS–PAGE Fernbach flasks (2 L) Freezer (−20˚C) Ice Incubator (37˚C) Rocker Screw-cap tubes (1.5 mL) Shaker (37˚C), for tubes and flasks Sonicator Spectrophotometer and cuvettes Toothpicks or wire loop, sterilized METHOD Bacterial Protein Expression This expression and purification procedure lasts 4 d, beginning with transformation and overnight incubation of competent bacterial cells. The transformed cells are used to begin starter cultures early on the second day, and
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078527
1095
D.C. Rio those cultures are later used to inoculate larger overnight cultures for protein expression. Lysis of the cells and protein purification takes place on day three, followed by a 24-h dialysis of the purified T7-RNAP.
1. Transform pT7-911Q into BL21-(DE3) competent cells. Plate on agar plates with LB medium and 50 µg/mL ampicillin. Incubate the plates overnight at 37˚C.
2. Prepare 900 mL of tryptone broth for each liter of culture by combining 10 g of tryptone, 5.5 g of NaCl, and 900 mL of ddH2O in a 2-L Fernbach flask. Autoclave for 20 min on a slow exhaust, liquid cycle. 3. Early the next day, prepare M9TB expression medium by adding the following to each flask of tryptone broth: 10× M9 salts 0.1 M CaCl2 1 M MgSO4 25% (v/v) glycerol 50 mg/mL ampicillin
100 mL 1 mL 1 mL 8 mL 0.8 mL
4. Start an inoculum by using a sterilized toothpick or wire loop to resuspend 2–3 fresh bacterial colonies (1 mm in diameter) in 1.0 mL of M9TB. Incubate the inoculum at 37˚C with shaking. It is important to use freshly transformed bacterial colonies obtained immediately after a transformation into BL21(DE3) competent cells. Do not store the plates at 4˚C.
5. At the end of the day, inoculate the Fernbach flasks. For each liter of M9ZB, use 100 µL of inoculum. Incubate the flasks overnight at 37˚C with shaking. 6. Check the OD600 of the cells in the flasks.
7. When the OD600 is 0.5–0.6, induce the cells in the flasks by adding 0.5 mL of 1 M IPTG per 1-L culture. 8. Continue to incubate the cells for 3 h at 37˚C with shaking.
9. Harvest the cells: in a Sorvall RC-5B centrifuge or equivalent, centrifuge the culture for 10 min in 500-mL bottles at 4000 rpm in a GS3 rotor or in 250-mL bottles at 7000 rpm in an SLA1500 rotor. Bacterial Lysis and Nickel-Agarose Affinity Purification
10. Resuspend the cells in 25 mL of lysis buffer per pellet. Transfer the cells to 50-mL conical tubes. 11. Add 1.0 mL of 10 mg/mL lysozyme, 50 µL of 20 mg/mL PMSF, and 20 µL of 5 mg/mL leupeptin. 12. Incubate the cells with rocking for 20 min at 4˚C.
13. Add 0.25 mL of 8% (w/v) sodium deoxycholate and incubate with rocking for 20 min at 4˚C.
14. Sonicate each pellet four times for 15 sec until dispersed. Keep the samples in ice during sonication. 15. Transfer the samples to 50-mL Oak Ridge centrifuge tubes. Centrifuge in a Sorvall SS-34 rotor at 15,500 rpm for 30 min at 4˚C. 16. Decant the supernatant into a clean bottle or flask, and keep it on ice.
17. Make a column of Ni-NTA agarose, using 10 mL of 50% slurry for each liter of starting cell culture. For example, for 2 L of cell culture, use 20 mL of 50% slurry.
18. Equilibrate the resin with ≥10 column volumes of lysis buffer. 19. Load the protein sample by gravity flow onto the column, and collect the flowthrough. 20. Wash the resin with three column volumes of lysis buffer followed by 10 column volumes of wash buffer. Collect the flowthrough. To remove all bacterial nuclease contamination from the purified protein, it is critical that you wash the column extensively with ≥10 column volumes of wash buffer before the imidazole elution.
1096
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078527
Preparation of Active T7 RNA Polymerase
21. Elute the T7-RNAP with 10 column volumes of elution buffer. Collect the eluent in aliquots of one column volume. 22. Check 5 µL of each fraction by SDS–PAGE on a 7.5% SDS-polyacrylamide gel. 23. Pool the peak fractions containing T7-RNAP (the protein runs at 100 kDa). 24. Dialyze the peak fractions against T7-RNAP storage buffer for 24 h at 4˚C, with three changes of 100 volumes each. 25. Quantitate the purified T7-RNAP by measuring the absorbance at 280 nm (A280). The estimated mg/mL = A280 × (dilution factor/1.3). Aliquot the enzyme in 100- to 200-μL aliquots into 1.5mL screw-cap tubes on ice in a cold room. 26. Store the purified T7-RNAP at −20˚C.
27. Determine the activity of the purified T7-RNAP. A practical way to determine activity is to perform in vitro transcription reactions with serially diluted enzyme from your preparation versus several amounts of commercially available enzyme; assay the incorporation of [α-32P]UTP by TCA precipitation or DEAE filter retention.
RELATED INFORMATION
The protein expression system described here is derived from the T7 promoter-driven system originally developed by Studier et al. (1990).
RECIPES Ni-NTA Elution Buffer
Reagent Ni-NTA lysis buffer (5×) β-Mercaptoethanol (14.1 M) Imidazole (1 M, pH 8.0) ddH2O
Quantity (for 200 mL)
Final concentration
40 mL 71 µL, add fresh 100 mL to 200 mL
1× 5 mM 500 mM
Ni-NTA Lysis Buffer
Reagent β-Mercaptoethanol (14.1 M) Imidazole (1 M, pH 8.0) ddH2O
Quantity (for 200 mL)
Final concentration
71 µL, add fresh 200 µL to 200 mL
5 mM 1 mM (pH 8.0)
Ni-NTA Lysis Buffer (5×)
Reagent Tris-HCl (1 M, pH 8.0) NaCl (5 M) Glycerol (100%)
Quantity (for 200 mL)
Final concentration (5×)
50 mL 20 mL 50 mL
250 mM 500 mM 25% (v/v)
Autoclave. Store for up to 6 mo at 4˚C. Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078527
1097
D.C. Rio
Ni-NTA Wash Buffer
Reagent Ni-NTA lysis buffer (5×) β-Mercaptoethanol (14.1 M) Imidazole (1 M, pH 8.0) ddH2O
Quantity (for 500 mL)
Final concentration
100 mL 177.5 µL, add fresh 5 mL to 500 mL
1× 5 mM 10 mM
T7-RNAP Storage Buffer
Reagent Potassium phosphate (0.2 M, pH 7.5) NaCl (5 M) Glycerol (100%) Dithiothreitol (DTT, 0.5 M) EDTA (0.25 M, pH 8.0) NaN3 powder (Sigma-Aldrich) ddH2O
Quantity (for 1 L)
Final concentration
100 mL 20 mL 500 mL 20 mL 0.4 mL 2g to 1 L
20 mM 100 mM 50% (v/v) 10 mM 0.1 mM 0.2% (w/v)
REFERENCES Ichetovkin IE, Abramochkin G, Shrader TE. 1997. Substrate recognition by the leucyl/phenylalanyl-tRNA-protein transferase. Conservation within the enzyme family and localization to the trypsin-resistant domain. J Biol Chem 272: 33009–33014.
1098
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185: 60–89.
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot078527
