TITLE: Isolation of ribosome-associated nascent chains of soluble proteins produced in Escherichia coli
AUTHORS: Renuka Kudva, Andreas Vogt, Kärt Denks, Gunnar von Heijne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Isolation of ribosome-nascent chains of co-translationally folded domains. Detailed description of protocol published in </div><div class = "text-block"><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1073/pnas.1810523115</span></a><span>. The arrest sequence used for stalling was TnaC (the leader peptide of the tryptophanase operon). </span></div></div>

[STEPS]
?. Expression tests (perform after transformation of constructs into KC6 cells)Day 1Pick 5 single clones from plates and seed into 2 ml LB supplemented with antibiotic of choice. Grow overnight at 37°C by shaking at 200 rpm. Prepare a master plate seeded with each of the clones used. Day 2 Sub-culture the overnight cultures into 50 ml of LB supplemented with antibiotics to an A600 of 0.1. Culture at 37°C till it reaches an A600 of 0.5. (Check A600 every 30 min). After cell growth to an A600 of 0.5, add 0.2% Arabinose (final concentration) to the cultures to induce expression and culture at   for   Cool cultures on ice for  Collect cells by centrifugation     at  Resuspend each cell pellet in   buffer A.Transfer the   of cell suspensions into several 2 ml reaction tubes. Supplement with 10mg/ml lysozyme and 10 mg/ml DNAse1. Lyse cells by repeated cycles of freezing and thawing. (Plunge reaction tubes into liquid nitrogen to freeze and transfer tubes to a thermoblock at 42°C to thaw. Repeat 10 times and mix the lysate well between cycles of freezing and thawing. Spin down lysate for   at   in a cooled table-top centrifuge at   . Transfer the lysate to ultracentrifuge tubes (polycarbonate tubes suited for the Beckman TLA 100.3 ultracentrifuge), underlie with Buffer B using a syringe and needle carefully to avoid bubbles. (1 ml of lysate and 1 ml of Buffer B per tube). Centrifuge at    for    in a TLA 100.3 rotor.Discard supernatant after centrifugation the ribosome go into the pellet.Resuspend ribosomal pellet in 30 ul buffer C. Measure concentration on a nanodrop/spectrophotometer at A260. Load different dilutions on SDS-PAGE (A260 of 7 for coomassie and A260 of 14 for Western blotting). Check for differences in expression and make a reserach cell bank (glycerol stocks) of the best expressing clone.After cell growth to an A600 of 0.5, add 0.2% Arabinose (final concentration) to the cultures to induce expression and culture at  for Cool cultures on ice for Collect cells by centrifugation  at Resuspend each cell pellet in  buffer A.Transfer the  of cell suspensions into several 2 ml reaction tubes. Supplement with 10mg/ml lysozyme and 10 mg/ml DNAse1. Lyse cells by repeated cycles of freezing and thawing. (Plunge reaction tubes into liquid nitrogen to freeze and transfer tubes to a thermoblock at 42°C to thaw. Repeat 10 times and mix the lysate well between cycles of freezing and thawing. Spin down lysate for   at   in a cooled table-top centrifuge at   . Transfer the lysate to ultracentrifuge tubes (polycarbonate tubes suited for the Beckman TLA 100.3 ultracentrifuge), underlie with Buffer B using a syringe and needle carefully to avoid bubbles. (1 ml of lysate and 1 ml of Buffer B per tube). Centrifuge at    for    in a TLA 100.3 rotor.Discard supernatant after centrifugation the ribosome go into the pellet.Resuspend ribosomal pellet in 30 ul buffer C. Measure concentration on a nanodrop/spectrophotometer at A260. Load different dilutions on SDS-PAGE (A260 of 7 for coomassie and A260 of 14 for Western blotting). Check for differences in expression and make a reserach cell bank (glycerol stocks) of the best expressing clone.Spin down lysate for  at  in a cooled table-top centrifuge at  . Transfer the lysate to ultracentrifuge tubes (polycarbonate tubes suited for the Beckman TLA 100.3 ultracentrifuge), underlie with Buffer B using a syringe and needle carefully to avoid bubbles. (1 ml of lysate and 1 ml of Buffer B per tube). Centrifuge at    for    in a TLA 100.3 rotor.Discard supernatant after centrifugation the ribosome go into the pellet.Resuspend ribosomal pellet in 30 ul buffer C. Measure concentration on a nanodrop/spectrophotometer at A260. Load different dilutions on SDS-PAGE (A260 of 7 for coomassie and A260 of 14 for Western blotting). Check for differences in expression and make a reserach cell bank (glycerol stocks) of the best expressing clone.Centrifuge at   for   in a TLA 100.3 rotor.Discard supernatant after centrifugation the ribosome go into the pellet.Resuspend ribosomal pellet in 30 ul buffer C. Measure concentration on a nanodrop/spectrophotometer at A260. Load different dilutions on SDS-PAGE (A260 of 7 for coomassie and A260 of 14 for Western blotting). Check for differences in expression and make a reserach cell bank (glycerol stocks) of the best expressing clone.If expression is not sufficient, check more clones or improve expression conditions.
37 °C
4 °C
5 ml
5 ml
4 °C
?. Purification of ribosome-associated nascent chains of soluble proteinsDay 1Prepare 2.1 litres of lysogeny broth (use high purity yeast extract and tryptone as described in the Methods section). I divided the medium into four flasks of 500 ml each. Prepare one flask with 100 ml for the seed culture. Autoclave. Prepare buffers.Seed the best expressing clone into 100 ml LB supplemented with antibiotic. Grow overnight at 37°C. Day 2Dilute the overnight cultures into the flasks containing 500 ml of LB (supplemented with antibiotic for selection) to an A600 of 0.1. Culture at 37°C to an A600 of 0.5. Check for growth every   Induce expression of plasmid with 0.2% arabinose for 1 hour. Chill cultures on ice for 30 min. Collect cells by centrifugation   for   at   in a JLA 8.1000 rotor. Measure cell mass (wet weight of the pellet). Resuspend cells in 2 ml BufferA/g cell mass.Lyse cells with an emulsifex (3 passes at 8000 psi). Clarify the cell lysate by centrifugation (  for   at  ) in a JA25-50 rotor. Distribute the cell lysate into Ti70 ultracentrifuge tubes (Beckman Coulter) (fill to around   ) and underlie with   of buffer B with a needle and syringe. Centrifuge at   for   at 4°C in a Ti70 rotor. Day 3Discard supernatant and gently resuspend the ribosome pellet in Buffer A (  ). Keep on ice and shake gently till the pellet goes into solution.While the pellet is on ice, prepare the Talon resin. Swirl the bottle to resuspend the resin, pipette out 1 ml in a 15 ml tube and wash thrice with 10 bed volumes of buffer A. Supplement the last wash with 10 ug/ml total E.coli tRNA. Transfer the ribosomal suspension to a dounce homogeniser and use the loose piston to homogenise. Transfer the homogenate to the washed Talon resin and incubate for    for binding (rotate the tube on a roller). Centrifuge at   for 2 min, discard supernatant.Wash with 5x 10 bed volumes of wash buffer.Elute with 2 bed volumes of elution buffer (incubate for    to    min with elution buffer) before collecting the elution.Transfer eluant to polycarbonate ultracentrifuge tubes suited for the Beckman Coulter TLA 100.3 rotor, and spin at   for   . Discard the supernatant by pipetting out immediately, and resuspend the 70S pellet obtained in   buffer C. (I recommend a small volume to have a concentrated sample that can be easily diluted for further use. Make small aliquots since it's best to avoid multiple freeze-thaw cycles). Resuspension to be carried out by adding requisite volume to the pellet and letting the pellet go into solution. Plunge freeze in liquid nitrogen and store at -80 °C long-term.Day 2Dilute the overnight cultures into the flasks containing 500 ml of LB (supplemented with antibiotic for selection) to an A600 of 0.1. Culture at 37°C to an A600 of 0.5. Check for growth every Induce expression of plasmid with 0.2% arabinose for 1 hour. Chill cultures on ice for 30 min. Collect cells by centrifugation   for   at   in a JLA 8.1000 rotor. Measure cell mass (wet weight of the pellet). Resuspend cells in 2 ml BufferA/g cell mass.Lyse cells with an emulsifex (3 passes at 8000 psi). Clarify the cell lysate by centrifugation (  for   at  ) in a JA25-50 rotor. Distribute the cell lysate into Ti70 ultracentrifuge tubes (Beckman Coulter) (fill to around   ) and underlie with   of buffer B with a needle and syringe. Centrifuge at   for   at 4°C in a Ti70 rotor. Day 3Discard supernatant and gently resuspend the ribosome pellet in Buffer A (  ). Keep on ice and shake gently till the pellet goes into solution.While the pellet is on ice, prepare the Talon resin. Swirl the bottle to resuspend the resin, pipette out 1 ml in a 15 ml tube and wash thrice with 10 bed volumes of buffer A. Supplement the last wash with 10 ug/ml total E.coli tRNA. Transfer the ribosomal suspension to a dounce homogeniser and use the loose piston to homogenise. Transfer the homogenate to the washed Talon resin and incubate for    for binding (rotate the tube on a roller). Centrifuge at   for 2 min, discard supernatant.Wash with 5x 10 bed volumes of wash buffer.Elute with 2 bed volumes of elution buffer (incubate for    to    min with elution buffer) before collecting the elution.Transfer eluant to polycarbonate ultracentrifuge tubes suited for the Beckman Coulter TLA 100.3 rotor, and spin at   for   . Discard the supernatant by pipetting out immediately, and resuspend the 70S pellet obtained in   buffer C. (I recommend a small volume to have a concentrated sample that can be easily diluted for further use. Make small aliquots since it's best to avoid multiple freeze-thaw cycles). Resuspension to be carried out by adding requisite volume to the pellet and letting the pellet go into solution. Plunge freeze in liquid nitrogen and store at -80 °C long-term.Collect cells by centrifugation  for  at  in a JLA 8.1000 rotor. Measure cell mass (wet weight of the pellet). Resuspend cells in 2 ml BufferA/g cell mass.Lyse cells with an emulsifex (3 passes at 8000 psi). Clarify the cell lysate by centrifugation (  for   at  ) in a JA25-50 rotor. Distribute the cell lysate into Ti70 ultracentrifuge tubes (Beckman Coulter) (fill to around   ) and underlie with   of buffer B with a needle and syringe. Centrifuge at   for   at 4°C in a Ti70 rotor. Day 3Discard supernatant and gently resuspend the ribosome pellet in Buffer A (  ). Keep on ice and shake gently till the pellet goes into solution.While the pellet is on ice, prepare the Talon resin. Swirl the bottle to resuspend the resin, pipette out 1 ml in a 15 ml tube and wash thrice with 10 bed volumes of buffer A. Supplement the last wash with 10 ug/ml total E.coli tRNA. Transfer the ribosomal suspension to a dounce homogeniser and use the loose piston to homogenise. Transfer the homogenate to the washed Talon resin and incubate for    for binding (rotate the tube on a roller). Centrifuge at   for 2 min, discard supernatant.Wash with 5x 10 bed volumes of wash buffer.Elute with 2 bed volumes of elution buffer (incubate for    to    min with elution buffer) before collecting the elution.Transfer eluant to polycarbonate ultracentrifuge tubes suited for the Beckman Coulter TLA 100.3 rotor, and spin at   for   . Discard the supernatant by pipetting out immediately, and resuspend the 70S pellet obtained in   buffer C. (I recommend a small volume to have a concentrated sample that can be easily diluted for further use. Make small aliquots since it's best to avoid multiple freeze-thaw cycles). Resuspension to be carried out by adding requisite volume to the pellet and letting the pellet go into solution. Plunge freeze in liquid nitrogen and store at -80 °C long-term.Clarify the cell lysate by centrifugation ( for  at ) in a JA25-50 rotor. Distribute the cell lysate into Ti70 ultracentrifuge tubes (Beckman Coulter) (fill to around  ) and underlie with  of buffer B with a needle and syringe. Centrifuge at  for  at 4°C in a Ti70 rotor. Day 3Discard supernatant and gently resuspend the ribosome pellet in Buffer A ( ). Keep on ice and shake gently till the pellet goes into solution.While the pellet is on ice, prepare the Talon resin. Swirl the bottle to resuspend the resin, pipette out 1 ml in a 15 ml tube and wash thrice with 10 bed volumes of buffer A. Supplement the last wash with 10 ug/ml total E.coli tRNA. Transfer the ribosomal suspension to a dounce homogeniser and use the loose piston to homogenise. Transfer the homogenate to the washed Talon resin and incubate for    for binding (rotate the tube on a roller). Centrifuge at   for 2 min, discard supernatant.Wash with 5x 10 bed volumes of wash buffer.Elute with 2 bed volumes of elution buffer (incubate for    to    min with elution buffer) before collecting the elution.Transfer eluant to polycarbonate ultracentrifuge tubes suited for the Beckman Coulter TLA 100.3 rotor, and spin at   for   . Discard the supernatant by pipetting out immediately, and resuspend the 70S pellet obtained in   buffer C. (I recommend a small volume to have a concentrated sample that can be easily diluted for further use. Make small aliquots since it's best to avoid multiple freeze-thaw cycles). Resuspension to be carried out by adding requisite volume to the pellet and letting the pellet go into solution. Plunge freeze in liquid nitrogen and store at -80 °C long-term.Transfer the homogenate to the washed Talon resin and incubate for   for binding (rotate the tube on a roller). Centrifuge at  for 2 min, discard supernatant.Wash with 5x 10 bed volumes of wash buffer.Elute with 2 bed volumes of elution buffer (incubate for    to    min with elution buffer) before collecting the elution.Transfer eluant to polycarbonate ultracentrifuge tubes suited for the Beckman Coulter TLA 100.3 rotor, and spin at   for   . Discard the supernatant by pipetting out immediately, and resuspend the 70S pellet obtained in   buffer C. (I recommend a small volume to have a concentrated sample that can be easily diluted for further use. Make small aliquots since it's best to avoid multiple freeze-thaw cycles). Resuspension to be carried out by adding requisite volume to the pellet and letting the pellet go into solution. Plunge freeze in liquid nitrogen and store at -80 °C long-term.Elute with 2 bed volumes of elution buffer (incubate for   to   min with elution buffer) before collecting the elution.Transfer eluant to polycarbonate ultracentrifuge tubes suited for the Beckman Coulter TLA 100.3 rotor, and spin at  for  . Discard the supernatant by pipetting out immediately, and resuspend the 70S pellet obtained in  buffer C. (I recommend a small volume to have a concentrated sample that can be easily diluted for further use. Make small aliquots since it's best to avoid multiple freeze-thaw cycles). Resuspension to be carried out by adding requisite volume to the pellet and letting the pellet go into solution.
4 °C
4 °C
12 ml
12 ml
5 ml
20 µl