Protein Production and Purification 124

Sf9 cells at 1.5-2 x 106/ml were infected with recombinant baculovirus, and harvested by centrifugation after 3 days. Sf9 cells expressing histidine-tagged TrkA498-796 _{(~7 liters of}

medium) were lysed by sonication in 100 ml of 50 mM NaKPO4, pH 8.0, containing 300

mM NaCl, 5% glycerol, 10 mM imidazole, 10 mM 2-mercaptoethanol, 0.5 mM PMSF and protease inhibitor cocktail (Roche). The lysate was then mixed with Ni-NTA beads (Qiagen) for 1 hour at 4°C. Beads were washed in 50 column-volumes of lysis buffer (described above), and bound TrkA-TKD was eluted with increasing concentrations of imidazole in 25 mM MES, pH 6, containing 300 mM NaCl, 5% (w/v) glycerol, 10 mM 2- mercaptoethanol, 0.5 mM PMSF and protease inhibitor cocktail (Roche). Eluted protein

was then further purified using a Fractogel SO3- cation exchange column (EMD)

equilibrated with 25 mM MES, pH 6, containing 5% glycerol, 2 mM DTT and eluting with a gradient from 10 mM to 1 M NaCl. TrkA was then applied to a HiTrap Butyl Sepharose HP column (GE Healthcare) in 25 mM MES, pH 6, containing 150 mM NaCl and 2 mM DTT, eluting with a gradient from 0.8 M to 0 M (NH4)2SO4, and subjected to a final step

of size exclusion chromatography using a Superdex 200 column (GE Healthcare) equilibrated in 25 mM MES, pH 6, containing 250 mM NaCl and 2 mM DTT.

Sf9 cells expressing histidine-tagged Ror2 TKD (~8 liters of medium) were lysed by sonication in 150 ml of lysis buffer, composed of 20 mM NaKPO4, pH 8.0, containing 200

mM NaCl, 10 mM 2-mercaptoethanol, 1 mM PMSF, 10 µM benzamidine, 2.3 µM

leupeptin, 2 µM aprotinin, and 3 µM pepstatin (Sigma). Cell lysates containing Ror2 TKD protein were mixed with Ni-NTA beads (Qiagen) for 30 minutes at 4°C, which were then washed with lysis buffer prior to elution of protein in lysis buffer containing 200 mM imidazole. Eluted protein was passed through a Fractogel TMAE column (EMD)

equilibrated with 25 mM Tris-HCl, pH 8, containing 100 mM NaCl and 2 mM DTT to remove anionic contaminants, and was then passed through a CHT2.1 hydroxyapatite

column (Bio-Rad) equilibrated in 20 mM HEPES, pH 8, containing 2.5 mM NaKPO4, 200

mM NaCl, 2 mM DTT, and 1 mM PMSF, prior to loading on to a HiTrap Butyl Sepharose HP column (GE Healthcare) in 25 mM Tris-HCl, pH 8, containing 125 mM NaCl and 2 mM DTT. Ror2-TKD was eluted from butyl sepharose with a gradient from 0.5 M to 0 M (NH4)2SO4 in this same buffer, and then subjected to size exclusion chromatography

using a Superdex 200 column (GE Healthcare) equilibrated in 20 mM Tris-HCl, pH 7.5,

The purification scheme described above for TrkA-TKD was used for the crystallization studies and initial autophosphorylation assays. However, the purification scheme was optimized for further studies of TrkA and TrkB TKDs due to heterogeneity of post translation modification and to ensure no phosphatase contamination. The cells were lysed and bound to Ni-NTA beads as described above for TrkA-TKD. However, the bound TrkAor TrkB TKD was eluted with increasing concentrations of imidazole in 50

mM NaKPO4, pH8, containing 300 mM NaCl, 5% (w/v) glycerol, 10 mM 2-

mercaptoethanol, 0.5 mM PMSF and protease inhibitor cocktail (Roche). Eluted protein was then further purified using a Fractogel SO3- cation exchange column (EMD)

equilibrated with 25 mM MES, pH 6, containing 5% glycerol, 2 mM DTT and eluting with a gradient from 10 mM to 1 M NaCl. TrkA and TrkB TKD proteins were concentrated to ~ 5-6 mls and treated with YopH phosphatase at 2 µM for 1 hour at 30° in the presence of 1mM EDTA. Pilot experiments with YopH had lower activity at a higher pH an

observation previously reported. Furthermore, EDTA was added during the phosphatase treatment since protein tyrosine phosphatase activity is not dependent on metals and in some cases metal ions can inhibit their activity. (Lu and Zhu, 2011; Zhang et al., 1992; Zhang, 2003). YopH was purified away using a Fractogel SO3- cation exchange column

(EMD) equilibrated with 25 mM HEPES, pH 8, containing 5% glycerol, 2 mM DTT. YopH is highly basic and binds very effectively to the cation exchange column at pH 8 where as both TrkA and TrkB TKDs do not bind. TrkA and TrkB TKD proteins were then concentrated to a volume of ~5-6 ml and treated with 2 µM λ phosphatase in the presence of 5 mM MnCl2 for 1 hour at 30°. Pilot experiments and previous reports

showed greater activity of λ phosphatase at pH 8 and with MnCl2 (Barik, 1993; Zhuo et al.,

column (EMD) equilibrated with 25 mM MES, pH 6, containing 5% glycerol, 2 mM DTT and eluting with a gradient from 10 mM to 1 M NaCl. TrkA or TrkB TKD was then applied to a HiTrap Butyl Sepharose HP column (GE Healthcare) in 25 mM MES, pH 6, containing 150 mM NaCl and 2 mM DTT, eluting with a gradient from 0.8 M to 0 M (NH4)2SO4, and subjected to a final step of size exclusion chromatography using a

Superdex 200 column (GE Healthcare) equilibrated in 25 mM Hepes, pH 8, containing 150 mM NaCl and 2 mM DTT.

YopH BL21 cells were grown in 1 liter of LB with 50 µg/ml streptomyocin at 37°C until a density of ~0.6 OD600 nm. At that point, the cells were cooled down to 18°C and induced

with 1mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown overnight. Cells were harvested by centrifugation at 2,000 x g for 15 minutes at 4°C. Cells were lysed by sonication in 50 mM Hepes, pH 7.4, 300 mM NaCl, 10% glycerol, 10 mM imidazole, 10 mM 2-mercaptoethanol, 0.5 mM PMSF and protease inhibitor cocktail (Roche). Protein lysate was incubated with Ni-NTA beads (Qiagen) for 1 hour at 4°C and then washed with 20 mM imidazole prior to eluting with 300 mM imidazole. YopH was then loaded on to a Fractogel SO3- cation exchange column (EMD) equilibrated with 25 mM MES, pH 6,

containing 5% glycerol, 2 mM DTT and eluting with a gradient from 10 mM to 1 M NaCl. Fractions corresponding to the peak were pooled, concentrated and loaded on to a size exclusion Superdex 200 column (GE Healthcare) equilibrated in 25 mM MES, pH 6, containing 250 mM NaCl and 2 mM DTT.

λ phosphatase was expressed and purified essentially as described (Zhuo et al., 1993). However, the first purification step was phenyl-sepharose chromatography and not Bio- Gel A chromatography. Instead, after the phenyl-sepharose column, the λ phosphatase fractions were pooled, concentrated and loaded on to a size exclusion (Superdex 200)

column (GE Healthcare) equilibrated in 25 mM Tris, pH 7.5, containing 150 mM NaCl. Purified λ phosphatase was stored at -80°C at 20 mg/ml in the buffer used for size exclusion chromatography plus a final concentration of 10% glycerol.