EL PLAN ESTRATÉGICO DE MARKETING

Cell resuspensions were thawed at 4 °C and pro tease inhibitors (5 mM benzamidine, 5 mM EDTA) were added to prevent degradation o f target proteins. Cells were lysed by two high pressure (1000 p.s.i.) passages through a cold (4-10 °C) French Pressure Cell Press (American Instrument Company) and by three consecutive cycles o f 30 seconds in a sonicator (MSE Soniprep 150, using a 150 mm probe, 8mM tip diameter, power rating 12-18 amplitude microns). Insoluble cell debris was removed from target protein by centrifugation (Sorvall RC-5B centrifuge, SS34 rotor, 15000 r.p.m. for 60 minutes at 4 °C). The GST-fusion protein was captured from the supernatant by incubation with glutathione-sepharose beads (Pharmacia) for 1 hour at 4 °C. The beads were washed extensively by three cycles o f centrifugation (Heraeus Labofuge 400R centrifuge, rotor type 8179, 1600 r.p.m. for 10 minutes) followed by resuspension in 10 ml 50 mM Tris pH 7.5, 50 mM NaCl, 5 mM benzamidine, 0.02% NaN^, 2 mM DTT buffer, per 1 ml slurry o f beads. The glutathione-sepharose beads plus GST-fusion protein were exchanged into 40 mM Tris pH 7.9, 10 mM NaCl, 10 mM CaClz, 6 mM MgCl^ buffer for treatment with D Nase I (Sigma) at 20 units per 1 mg GST-fusion protein by incubation for 60 minutes at 25 °C. DNase I was removed by a further buffer exchange step. At this point there follow two alternative purification strategies depending the intended use o f the protein.

For the purposes o f the biosensor experiments, the D Nase I treated GST- fusion protein was eluted from the glutathione-sepharose beads by competition with 5 ml 50 mM Tris pH 8.0, 150 mM NaCl, 10-20 mM glutathione buffer. The GST- fusion protein was further purified, separated from glutathione and exchanged into a suitable buffer (50 mM Tris pH 7.5, 50 mM NaCl, 0.02% NaN^, 2 mM DTT) by preparative-scale gel filtration (Pharmacia XK-16 column. Superose-12 resin). The gel filtration elution fractions containing target protein were pooled and concentrated using centriprep and centricon (Amicon) concentrators with appropriate (3 kDa or 10 kDa) molecular weight cut-off membranes. The purified GST-fusion proteins were stable when stored for several weeks at 4 °C.

For the NMR, AUG and SEC experiments it was necessary to produce isolated target protein by thrombin digestion o f the linker peptide connecting the GST moiety and the target protein. For thrombin digestion, the beads with captured GST-fusion protein were exchanged into 50 mM Tris pH 8.0, 150 mM NaCl, 2.5 mM CaCl2 buffer, 5 ml per 1 ml slurry, and 1 unit thrombin (Calbiochem) per 1 mg fusion

protein was added with incubation at 25 °C for 2-4 hours. Cleavage was terminated by addition o f 5 mM benzamidine and the beads (plus bound GST) were separated from the released target protein by centrifugation (Heraeus centrifuge, 2500 r.p.m. for 10 minutes at 4 °C). The target protein was further purified and buffer exchanged by gel filtration (as above) into 50 mM Tris pH 7.5, 50 mM NaCl, 0.02% NaN^, 2 mM D TT buffer. Finally, the protein sample was exchanged into a buffer suitable for NMR (50 mM deuterated Tris (^H^-Tris from Cambridge Isotope Laboratories, Inc.) pH 7.5, 50 mM NaCl, 0.02% NaN^, 2 mM DTT, 10% (v/v) D2O) or for AUC and

SEC (50 mM Tris pH 7.5, 50mM NaCl, 0.02% NaNj, 5mM (3-mercaptoethanol). To avoid centrifugation for long periods, which can result in sample loss, these final buffer exchanges were performed on a 5 ml NAP-5 column (Pharmacia) containing Sephadex G-25 resin.

Throughout the purification procedure sample purity was monitored by polyacrylamide gel electrophoresis (PAGE) performed under denaturing conditions. The proteins are incubated at 95 °C with SDS which binds covalently along the length o f the denatured polypeptide chains, independent o f the amino acid sequence, in a ratio proportional to the molecular weight o f the protein (at saturation, 1.4 g SDS binds 1 g protein). The negatively charged SDS thus allows electrophoretic separation o f the proteins primarily according to their molecular weights. SDS-PAGE was performed in two consecutive polyacrylamide gels: an upper stacking gel and a lower resolving gel. Further details o f the SDS-PAGE procedure are given below.

Proteins ran through the stacking gel (5 % polyacrylamide, 130 mM Tris-HCl pH 6.8, 0.1% SDS, 0.1% ammonium persulphate (APS), 0.1% N ,N ,N ’,N ’- tetramethylethylenediamine (TEMED) as a tight band, achieving a sharp focusing o f the sample before reaching the resolving gel. The sample proteins became separated as they migrated through the resolving gel (8-18% polyacrylamide, 40 mM Tris-HCl pH 8.8, 0.1% SDS). A stock o f polyacrylamide containing 29.2% (w/v) acrylamide and 0.8% (w/v) N ,N ’-methylenebisacrylamide was used to prepare resolving gels o f 14-18% polyacrylamide for resolving proteins in the range 8-60 kDa, or 8-14%

polyacrylamide for resolving larger proteins o f 20-150 kDa. The gels (dimensions 80 mm X 70 mm and 0.75 mm thickness) were poured immediately after enabling polymerisation by introducing the APS and TEM ED. The wells for loading protein samples onto the gels were made by inserting a 1 0 or 15-tooth comb into the stacking

gel prior to complete polymerisation. Protein samples containing 1-10 pg protein were prepared for SDS-PAGE by incubation in 15 pi 50 mM Tris-HCl pH 6.8, 2%

SDS, 2% glycerol, 1% (3-mercaptoethanol, 0.1 % bromophenol blue buffer for 5 minutes at 95 °C. The samples were loaded under Tris-glycine electrophoresis buffer (25 mM Tris-HCl pH 8.3, 250 mM glycine, 0.1 % SDS) and run on a Mini-protean II kit (BIORAD) at 180 V (constant voltage) for 50-60 minutes.

Following SDS-PAGE, proteins were viewed by specific staining o f protein by incubating the gel in 50ml 45% methanol, 10% acetic acid, 0.25% Coomassie Brilliant Blue R-250, 45% H2O for 30 minutes. Staining o f non-protein components

was removed by incubating the gel in 50 ml 20% methanol, 7% acetic acid, 73% HjO. An estimation o f the sample protein sizes was made by comparison with protein standards (R & D Systems Europe) run as markers on each gel.

The concentration o f essentially homogeneous protein was determined by UV-spectroscopy (p — 280 nm) and applying the Beer-Lambert Law (Campbell and Dwek, 1984) using a value for the molar extinction coefficient determined empirically by a established method (Gill and Von Hippel, 1989). The GST gene-hision system allowed variable yields for the different proteins, ranging from 2 - 2 0 mg purified target

protein per litre o f culture medium grown. A typical set o f biosensor assays required only 1-2 mg purified protein, whereas NMR samples required 5-20 mg purified protein in 600 pi (approximately equivalent to a concentration o f 0.5-2.0 mM for a 15 kDa protein).

For heteronuclear NMR studies it was necessary to produce ^^N-labelled target protein. Expression and purification o f uniformly ^^N-labelled protein was achieved as for the unlabelled protein produced, described above, except that following the initial overnight growth o f a single colony in 5 ml LB (Amp), bacterial cultures were grown in a vitamin-supplemented minimal M9 medium with (^^NH4)2S0 4 as the sole nitrogen source, as described previously (Clore et al., 1991)

(Ikura et al., 1991). Established M9 media (Sambrook et al., 1989) were used as a basis from which a new M9 medium was developed. The improved M9 medium contains a

dissolved commercially available vitamin tablet (Centrum, Whitehall) which was found to improve bacterial growth rates.

The methods described above were applicable to all the GST-fusion proteins used during these studies. One notable modification was that the SH3-BCR protein required an additional step o f purification after thrombin cleavage and elution from the glutathione-sepharose beads. In this case, the SH3-BCR protein was purified by ion-exchange chromatography using a cation exchange column (Perseptive Biosystems, POROS 20HS) equilibrated in 50 mM Tris pH 7.5, 50 mM NaCl, 0.02% NaNj, 2 mM DTT buffer. Bound SH3-BCR protein was eluted with a 50-500 mM NaCl gradient. Fractions containing SH3-BCR were pooled and the purification continued with gel filtration, as described above.