RESULTADOS RESPECTO A LOS OBJETIVOS

Hydrophobic interaction chromatography was carried out using a FPLC system m machine (Pharmacia, Uppsala, Sweden). Pooled fractions from the Anion-Exchange chromatography stage (section 2.2.4.7) were made up to 1.2M ammonium sulphate and loaded onto a ImL HiTrap™ Phenyl Sepharose HP column (small scale) or a 20mL Phenyl Sepharose HP column (large scale) pre-equilibrated with Buffer B (2.1.4.8) containing IM ammonium sulphate. Protein was eluted from the column by decreasing the ammonium sulphate concentration to OM at a flow rate of 1.0 mL/min over 40mL (small scale) or 480mL (large scale). LOmL fractions (small scale) or 5.0 mL fractions (large scale) were collected and stored at 4°C for subsequent analysis.

2.2.4.9 Concentration of the AmlC/AmlR complex by ultrafiltration

Following the analysis of fractions from hydrophobic interaction chromatography (section 2.2.4.8) pooled fractions containing most AmiC/AmiR were concentrated to approximately 16 mg/mL using a PM30 Diaflo ultrafilter (30,000 MW cut-off) in an ultrafiltration cell, both manufactured by Amicon Inc., Beverly, MA, USA. The final AmiC/AmiR complex purification was by analytical gel filtration step (see section 2.2.4.10).

2.2.4.10 Analytical gel filtration

Analytical gel filtration was carried out using an FPLC system 111 machine (Pharmacia, Uppsala, Sweden). Small protein samples not exceeding 200pL in volume or containing more than 5mg of protein were loaded onto a Superose 12 HR 10/30 column pre-equilibrated in the appropriate buffer. Separation was carried out in the same buffer and 200|iL fractions were collected and stored at 4°C or immediately concentrated for analysis (section 2.2.4.12) by TCA precipitation (section 2.2.4.11)

2.2.4.11 Protein concentration by Trichloroacetic acid (TCA) precipitation

A 10% (v/v) TCA solution was added to give a final concentration of 5% (v/v). The samples were mixed by vortexing and incubated at 4°C for 10 min. The precipitated protein was pelleted by centrifugation in an Eppendorf centrifuge at 14,000 rpm for 5 min at 4°C and the supernatant discarded. The pellets were washed in 200pL ice cold acetone by vortexing briefly and incubating at 4°C for 10 min. The protein was pelleted

by centrifugation in an Eppendorf centrifuge at 14,000 rpm for 5 min at 4°C and air dried before resuspension in 20pL protein loading buffer (2.1.4.6). Resuspended protein pellets were heated at 100 °C for 3 min and loaded onto SDS-Polyaciylamide gels.

2.2.4.12 SDS-Polyacrylamide gel electrophoresis

Analytical gel electrophoresis was carried out on 1.5mm thick 10-15% (v/v) polyacrylamide mini gels as described by Sambrook et al., 1989 using a Hoefer Tall Mighty Small 11cm vertical slab unit SE 280™. Samples were diluted 1:4 (v/v) with protein loading buffer (section 2.1.4.6) and heated at 100 °C for 3 mins prior to loading. The gels were electrophoresed in Ix Tris-glycine electrophoresis buffer (25mM Tris HCl, 250mM glycine, 0.1 (w/v) SDS) at a constant current of 20-30 mA for approximately 4 hours, or until the dye front had reached the bottom of the gel. Following electrophoresis, the stacking gel was removed and the resolving gel was stained with Coomassie Brilliant Blue R stain solution (45% (v/v) methanol, 10% (v/v) glacial acetic acid, 0.25% (w/v) Coomassie Brilliant Blue R stain) at room temperature for 1 hour. The gel was destained using destain solution (40% (v/v) methanol, 10% (v/v) glacial acetic acid) at room temperature until the protein bands could be clearly seen and there was no blue background.

2.2.4.13 W estern analysis

Proteins were separated by SDS-Polyacrylamide gels (section 2.2.4.12) and transferred onto Hybond-C extra nitrocellulose filters (Amersham) using a multiphor 11 electrophoresis system (LKB). Western blots (Harlow and Lane, 1988) were carried out for the detection of AmiR using a polyclonal antibody raised in rabbit against the AmiR- MBP fusion (Wilson et at. 1996).

A nitrocellulose filter, eight pieces of 3MM Whatmann filter paper (all cut to the same size as the SDS-polyacrylamide gel) and two green scourer pads were soaked in Transfer buffer (150 mM glycine, 25 mM Tris, 20% methanol) for a few minutes prior to protein transfer. The SDS-Polyacrylamide gel and the nitrocellulose membrane were sandwiched (avoiding air bubbles) in-between four pieces of 3MM Whatmann filter paper, a green scourer pad and a plastic plate on each side and placed into a tank filled

Chapter 2: MATERIALS & METHODS

with Transfer buffer so that the SDS-Polyacrylamide gel was on the side closest to the cathode. Electrophoresis was carried out at 50V for 75 to 90 min, after which the apparatus was dis-assembled and the nitrocellulose filter was placed in a tray containing 200mL of blocking solution (lOg of non-fat dried milk dissolved in PBS) at 4°C overnight. The nitrocellulose filter was placed in a heat sealable bag containing 15mL of blocking solution and a 1:1000 dilution of rabbit antisera raised against MalE-AmiR fusion protein, after which the bag was sealed avoiding as many air bubbles as possible and incubated at room temperature on a rotational shaker for 2 hours. The primary antibody solution was discarded and the nitrocellulose filter was placed in a tray containing PBS and washed by rotational shaking for 30 min. To ensure that all unbound antibody had been removed the PBS was replaced with fresh PBS after every 10 min. The nitrocellulose filter was placed into a heat sealable plastic bag containing 15mL of blocking solution and a 1:1000 dilution of horseradish peroxidase labelled sheep anti-rabbit IgG (Sigma). The bag was sealed avoiding as many air bubbles as possible and incubated at room temperature on a rotational shaker for 1 hour. The secondary antibody solution was discarded and the nitrocellulose filter was washed three times for 10 min in a tray containing PBS (as above). The colour was developed by addition of freshly prepared detection solution [83mL PBS, 17mL 4CN stock (150mg 4CN dissolved in 50mL methanol), 20pL hydrogen peroxide] by gentle shaking for 5 to

10 min. Developed blots were washed in distilled water and air dried. 2.2.5 Crystallisation methods

2.2.5.1 Concentration and buffer exchange of the AmiC/AmlR complex

Following the final analytical gel filtration stage, the AmiC/AmiR complex was concentrated down to approximately 50 mg/mL and buffer exchanged into crystallisation buffer (2.1.4.10) using Centricon 30 (MW cut-off = 30,000)(Amicon Inc., Beverly, MA, USA) concentrators by diluting out the original buffer 1000-fold. The concentrated AmiC/AmiR complex was used as a stock solution and diluted according to experimental requirements. The stock solution was stored at 4°C.

2.2.5.2 Micro-batch screen with AmiC/AmiR complex

Following purification, concentration and buffer exchange the AmiC/AmiR complex was tested using a crystallisation wild screen kit; CRYSTAL SCREEN™ (Hampton Research, Riverside, CA, USA) (Jancarik and Kim, 1991). l.OpL of AmiC/AmiR complex (approximately 10 mg/mL) was mixed with l.OpL of each of the 50 different conditions offered by CRYSTAL SCREEN™. The resulting 2.0pL drop was immersed under mineral oil in the well of a multi-well Terasaki plate (Greiner Labortechnik Ltd., Stonehouse, Glos., UK) and left undisturbed for approximately 1 week at 16°C to allow crystal growth.

2.2.5.3 Automated titration of Condition 40

Automated titration was carried out in a Terasaki plate under oil using a Lnpaxl Robot with Xstep software (Douglas Instruments Ltd., London, UK). Stock solutions were prepared as follows: 30% PEG 4000/2-Propanol, IM Sodium citrate pH 5.6. Stock solutions were diluted to give the following final concentrations: 5mg/mL AmiC/AmiR complex, PEG 4000/2-Propanol (variable), 50mM Sodium citrate pH 5.6. Results of the titration were optically checked using the microscope to determine and record if any precipitation had occurred. The Terasaki plates were left undisturbed for 2 to 5 days at

16°C to allow crystal growth.

2.2.5.4 Streak seeding with Condition 40

Conditions for streak seeding were prepared using the Impaxl Robot. A probe consisting of a brush hair permanently mounted on a glass capillary tube was brushed against a crystal and serially dipped into a saturated AmiC/AmiR complex solution. The probe was washed in distilled water after every set of conditions had been seeded. The Terasaki plates were left undisturbed for 2 to 5 days at 16°C to allow crystal growth.

2.2.5.5 Recovery and Mounting of crystals

Wells containing crystals were filled with lO.OjxL of recovery solution (2.1.5.4). Clumps of crystals were broken up using a probe. Crystals in recovery solution were transferred into a thin walled glass capillary tube (Glas, Berlin, Germany) using a pipette. Excess recovery solution was removed using filter paper strips so that the

Chapter 2: MATERIALS & METHODS

crystal remained stuck to the wall of the glass capillary with a minimum amount of solution surrounding it. Recovery solution was added to either end of the glass capillary. To keep the hydration of the crystal constant both ends of the glass capillary were sealed with wax.

Alternatively, crystals in recovery solution were transferred using a pipette onto a microscope coverslide containing approximately l.OmL of recovery solution on its surface. The crystal was observed under the microscope and ‘fished out* using a 200pm diameter loop (Hampton Research, Riverside, CA, USA).

2.2.5.6 In-house X-Ray data collection

Mounted crystals were fixed to the goniometer and positioned in the X-Ray pathway by viewing with a CCD camera connected to a video. Data was collected using a rotating anode generator (Rigaku RU200HB, Japan) producing CuK a radiation and the images were recorded on a Mar 300mm image plate (MARresearch, Germany). The angle and exposure time of data collection was varied according experimental requirements.

2.2.5.7 Freezing of crystals

Freezing was carried out using glycerol as the standard cryo-precipitant. Crystals were mounted onto loops (2.2.5.5) and serially immersed in Recovery solution (2.1.5.4) containing 12, 15, 20 and 25% glycerol instead of the 2-Propanol additive. Crystals were frozen in a stream of dry nitrogen at 1 lOK using the X-Stream cryo-cooling unit (Molecular Structure Corp., USA). Data was collected as described in section 2.2.5.6. 2.2.6 Computer Modeling methods

Models of the mutant AmiC proteins from PACl 11 and PAC153 were built using the program Modeller4 (Sali and Blundell, 1993). The models were analysed using the programs Procheck (Laskowski et oA, 1993), PROMOTIF (Hutchinson and Thornton, 1996), Naccess (Hubbard and Thornton, 1993) and Hbplus (McDonald and Thornton, 1994). Structures were superimposed onto the wild-type AmiC using the program ProFit (Martin, 1995). Visual inspection was performed using the program Insightll (Biosym/MSl, San Diego, CA, USA).

3. ANALYSIS OF AMIDASE REGULATORY