Supplemental Experimental Procedures

Transcription

1 Supplemental Experimental Procedures Cloning, expression and purification of untagged P C, Q D -N 0123, Q D- N 012 and Q D -N 01 - The DNA sequence encoding non tagged P C (starting at the Arginine 57 to Aspartate 235), Q D -N 0123 (starting at the Glutamate 35 to Alanine 335), Q D -N 012 (starting at the Glutamate 35 to Proline 275) and Q D -N 01 ) (starting at the Glutamate 35 to Histidine 208) was cloned into the Gateway petg-20a expression vector leading respectively to plasmid petg-20a-p C, petg-20a-q D -N 0123, petg-20a-q D -N 012 and petg-20a- Q D -N 01 ; a his6-tag encoding DNA and the sequence encoding for a TEV protease cleavage site were inserted in frame between the attb1 and the sequence encoding P C and Q D -N 0123, Q D -N 012 and Q D -N 01 (see table S1 for vectors and plasmids list). Primers (P C, Q D -N 0123, Q D -N 012 and Q D -N 01 - Forward and P C, Q D -N 0123, Q D -N 012 and Q D -N 01 -Reverse) used for amplification are shown in table S2. E. coli BL21 (DE3) plyss (Invitrogen) cells were transformed with petg-20a-p C, petg-20a-q D - N 0123, petg-20a-q D -N 012 and petg-20a- Q D -N 01. Precultures grown on Luria Broth at 37 C were used to start the culture (OD 600 = 0.4) at 37 C in TB medium. At OD 600 =0.8 the temperature was decreased to 25 C and the cells were cultivated for 18 hours. Ampicillin 100 µg/ml and Cloramphenicol 35 µg/ml were present continuously during expression. The purification of P C, Q D - N 0123, Q D -N 012 and Q D -N 01 started by a cell lysis step performed at 4 C. Cells were resuspended in lysis buffer (50 mm Tris ph 8.0, 300 mm NaCl, 1 mm EDTA, 0.5 mg/ml lysozyme, 1 mm phenylmethylsulfonyl fluoride (PMSF)), submitted to three freeze-thawing cycles and sonicated after the addition of DNase at 20 µg/ml and MgCl 2 at 20 mm. Pellet and soluble fractions were separated by centrifugation for 30 min at g. Soluble fractions containing P C, Q D -N 0123, Q D -N 012 and Q D - N 01 were: 1) dialyzed against 50 mm Tris, 150 mm NaCl, ph 8.0, 2) loaded on a Nickel (HisTrap FF crude Column cm (5 ml)) on ÄKTA Express (Amersham Biosciences) pre-equilibrated in 50 mm Tris, 300 mm NaCl, 10 mm Imidazole, ph 8.0, 3) eluted by washing with 50 mm Tris, 300 mm NaCl, ph 8.0 in the presence of 250 mm Imidazole, 4) desalted on HiPrep 26/10 Desalting column (SephadexTM G-25, Amersham Biosciences), 5) cleaved by the TEV protease 1mg/ml (4 C, 18 hours), 6) loaded on a Nickel column pre-equilibrated in 50 mm Tris, 300 mm NaCl, 10 mm Imidazole, ph 8.0, which selectively retard both the TEV and the fusion protein which are still his6- tagged. The untagged proteins (P C, Q D -N 0123, Q D -N 012 and Q D -N 01 ) were collected in the flow-through and concentrated on Centricon, cutoff 3 kda. After concentration, the proteins were purified on a Sephadex G75 equilibrated in 50 mm Phosphate, 150 mm NaCl, ph 7. Cloning, expression and purification of the his6-tagged P C ( His P C )- The DNA sequence encoding for his6-tagged periplasmic domain of XcpP C ( his P C ) was cloned into the Gateway pdest-17 expression vector leading to plasmid pdest-17- his P C (see table S1 for vectors and plasmids list). E. coli BL21 (DE3) plys S (Invitrogen) cells were transformed with pdest-17- his P C. Precultures grown on Luria Broth at 37 C were used to start the culture (OD 600 = 0.4) at 37 C in TB medium. At OD 600 =0.8 the temperature was decreased to 25 C and the cells were let grow for 18 hours. Ampicillin 100 µg/ml and Cloramphenicol 35 µg/ml were present continuously during expression. The purification of his P C started by a cell lysis step performed at 4 C. Cells were resuspended in lysis buffer (50 mm Tris ph 8.0, 300 mm NaCl, 1 mm EDTA, 0.5 mg/ml lysozyme, 1 mm phenylmethylsulfonyl fluoride (PMSF)), submitted to three freeze-thawing cycles and sonicated after the addition of DNase at 20 µg/ml and MgCl 2 at 20 mm. Pellet and soluble fraction were separated by centrifugation for 30 min at g. Soluble fractions containing his P C were: 1) dialyzed against 50 mm Tris, 150 mm NaCl, ph 8.0, 2) loaded on a Nickel (HisTrap FF crude Column cm (5 ml)) on ÄKTA Express (Amersham Biosciences) pre-equilibrated in 50 mm Tris, 300 mm NaCl, 10 mm Imidazole, ph 8.0, 3) eluted by washing with 50 mm Tris, 300 mm NaCl, ph 8.0 in the presence of 250 mm Imidazole, 4) The his6-tagged his P C in the eluted fractions were concentrated on Centricon, cutoff 3 kda and passed through a Sephadex G75 equilibrated in 50 mm Phosphate, 150 mm NaCl, ph 7. The final concentration of the proteins was calculated on the value of OD 280nm, by using the following extinction coefficients found by ProtParam (P C (mg/ml) -1, Q D -N (mg/ml) -1, Q D -N (mg/ml) -1 and Q D -N (mg/ml) -1, his P C (mg/ml) -1. Yields were 8, 7, 8, 15, 20 mg/l for P C, Q D -N 0123, Q D -N 012, Q D -N 01, his P C, respectively evaluated by NanoDrop (Thermo Scientific). 1

2 Batch co-purification (pull-down) of his P C and Q D -N 0123, Q D -N 012, Q D -N 01 and quaternary complex U H - V I -W J -X K periplasmic domains- We used the his P C periplasmic domain as bait. All experiments were performed at 4 C. In reaction mixture 1 (RM1), his P C was incubated in 1 ml of Equilibration Buffer (EqB) (50 mm TrisHCl/pH 8; 300 mm NaCl; 10 mm Imidazole) with 200 µl of 5% Ni-NTA magnetic bead solution (Qiagen) pre-equilibrated in EqB. In reaction mixture 2 (RM2), the untagged partners were incubated in 1 ml of EqB. Both RM1 and RM2 were placed on a rotary shaker and gently mixed for 1 hour. RM1 was placed on a magnet for 1 min to collect the magnetic beads. The flow-through (FT) was discarded, and the magnetic-beads were rinsed twice with 500 µl of EqB (W1 and W2), including a 1 min mix on the rotary shaker and a 1 min recovery on the magnet after each wash. Then, RM2 was mixed with the his N-coated magnetic beads from RM1 and gently mixed on the rotary shaker for 1 hour. To analyze this fraction, 18 µl of the 500 µl of the reaction mixture containing tagged and/or untagged proteins was mixed with 6 µl of 4X SDS-Loading buffer (Invitogen) and were boiled for 10 min and then loaded and run on a 15% SDS-PAGE (Fig 3A, C, D, E, line L). After a 1 min catch on the magnet, the flow-through (FT) was discarded, and the magnetic beads were washed with 500 µl of wash buffer (WB) (50 mm TrisHCl/pH 8, 300 mm NaCl, 20 mm Imidazole) height times (W1-W8), including a 1 min mix on the rotary shaker and a 1 min catch on the magnet for each wash. Proteins specifically bound to the magnetic beads were then eluted with 100 µl of elution buffer (EB) (50 mm TrisHCl/pH 8; 300 mm NaCl; 500 mm Imidazole) two times (E1-E2), including a 1 min mix on the rotary shaker, a spin at 8734 x g to pellet the beads and a 1 min catch on the magnet for each elution. To analyze each elution fractions, 18 µl of each protein sample was mixed with 6 µl of 4X SDS-loading buffer. The 24 µl samples were boiled for 10 min and then loaded and run on a 15% SDS-PAGE gel as described in. After electrophoresis, gels were stained with Coomassie Blue. For the co-purification experiment, we used 100 µg of his P C, 95 µg of Q D -N 0123, Q D -N 012 and Q D -N 01 and 90 µg of the U H -V I -W J -X K quaternary complex. 2

3 Supplemental tables Table S1: Vectors and plasmids used in this study Vectors & Plasmids pet-22b-gw pet-22b-gw-t G25-148NH Genotypea pelb, T7lac, Ap R, origin = pbr322, C-ter his6 tag, gateway site added for recombination cloning. attr1/attr2 987bp coding for the soluble domain of T G with a N-ter TEV protease cleavage site and a his6 tag, fused to the pelb cloned into the pet-22b Source or Reference (2) A. Geerlof, EMBL petg-20a T7/lac, Cam R, origin = pbr322, N-ter Trx-his6-tag petg-20a-x K30-333NH attr1/attr2 987bp coding for the soluble domain of X K with a N-ter petg-20a-w J28-237NH petg-20a-u H29-172NH petg-20a-v I28-129NH petg-20a-pc57-235nh petg-20a-q D -N NH petg-20a-q D -N NH petg-20a-q D -N NH pdest-17-gw attr1/attr2 708bp coding for the soluble domain of Wp with a N-ter attr1/attr2 510bp coding for the soluble domain of U H with a N-ter attr1/attr2 360bp coding for the soluble domain of V I with a N-ter TEV protease cleavage site fused to the Trx cloned into attr1/attr2 588bp coding for the soluble domain of P C with a N-ter TEV protease cleavage site fused to the Trx cloned into attr1/attr2 570bp coding for the soluble domain of Q D -N 01 with a N- ter TEV protease cleavage site fused to the Trx cloned into the petg- 20A attr1/attr2 771bp coding for the soluble domain of Q D -N 012 with a N- ter TEV protease cleavage site fused to the Trx cloned into the petg- 20A attr1/attr2 1008bp coding for the soluble domain of Q D -N 0123 with a N-ter TEV protease cleavage site fused to the Trx cloned into pelb, T7lac, ApR, origin = pbr322, N-ter his6 tag, gateway site added for recombination cloning. pdest-17- his P C attr1/attr2 588bp coding for the soluble domain of his P C with a N-ter TEV protease cleavage site fused tag-6-histidine cloned into the pdest-17 Invitrogen a Ap R, Cam R for Ampicilline and chloremphenicol resistance. Trx for Thioredoxin. 3

4 Table S2: Oligonucleotides used in this study Primer names T G -Forward T G -Reverse U H -Forward U H -Reverse V I -Forward V I -Reverse W J -Forward W J -Reverse X K -Forward X K -Reverse his P C / P C -Forward his P C /P C -Reverse Q D -N 01 -Forward Q D -N 01 -Reverse Q D -N 012 -Forward Q D -N 012 -Reverse Q D -N Forward Q D -N Reverse Primer sequences a 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTACACCACCACCACCACCACGAAAACCTGTACTTCCAGGGTATGAGCCGTCCCGACCAGGCCA 3 5 GGACCACTTTGTACAAGAAAGCTGGGTCTTATTAGTTGTCCCAGTTGCCGATGTCG 3 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTACACCACCACCACCACCACGAAAACCTGTACTTCCAGGGTAGCACCGGCTTCGCCAGCACCTC 3 5 GGACCACTTTGTACAAGAAAGCTGGGTC TTATTATCGCCGCGCCACCTCGACCCGTGG 3 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTACACCACCACCACCACCACGAAAACCTGTACTTCCAGGGTAGTGCCAGCGCGCGCAGCCTGC 3 end sequence 5 GGACCACTTTGTACAAGAAAGCTGGGTCTTATTATGGCTGGCTCCCGAGGAAACCGACGAGG 3 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTA CACCACCACCACCACCACGAAAACCTGTACTTCCAGGGTCGCATGTTCGACAGCGTGATGC 3 5 GGACCACTTTGTACAAGAAAGCTGGGTCTTATTATTCCGGCGCCCCCGGCATGCCTTCCGGC 3 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTACACCACCACCACCACCACGAAAACCTGTACTTCCAGGGTCGCCAGCAGTTGGCGATACGCAGCAGC 3 5 GGACCACTTTGTACAAGAAAGCTGGGTCTTATTATCGCTCGTCCTTCTTCCAATCGTCGCCG 3 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTA GAAAACCTGTACTTCCAGGGTCGCCTGCAACGCAGCCCGGTAGCCGTCGC 5 GGACCACTTTGTACAAGAAAGCTGGGTCTTATTAGTCGCTTTCCGTAGGCGTGGTCGTGGG3 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTA GAAAACCTGTACTTCCAGGGTCGCCTGCAACGCAGCCCGGTAGCCGTCGC 5 GGACCACTTTGTACAAGAAAGCTGGGTCTTATTAGTGGCTGCCCTTCTGGTCGAGCTGGCG 3 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTA GAAAACCTGTACTTCCAGGGTGAAAACAGCGGCGGGAACGCCTTCG 5 GGACCACTTTGTACAAGAAAGCTGGGTCTTATTACGGGGTGTCCAGCGATTGCGC 3 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTTA GAAAACCTGTACTTCCAGGGTGAAAACAGCGGCGGGAACGCCTTCG 5 GGACCACTTTGTACAAGAAAGCTGGGTCTTATTACGGCACGTCGAGCTGGCGAACGATGTCTTCG 3 a attb1 and recombination sites as well as the sequence coding for TEV protease recognition site are underlined and annotated; sequence encoding the 6his-tag is in bold 4

5 References 1. Douzi, B., Durand, E., Bernard, C., Alphonse, S., Cambillau, C., Filloux, A., Tegoni, M., and Voulhoux, R. (2009) J Biol Chem 284(50), Veesler, D., Blangy, S., Siponen, M., Vincentelli, R., Cambillau, C., and Sciara, G. (2009) Anal Biochem 388,