Prueba de Hipótesis especifica

Strains were grown up for Western blotting at least twice independently and the protein extract analysed and compared for consistency. Values for NitA are shown in brackets and NrtA is shown in the main text.

Aspergillus crude plasma membrane preparation

Crude membrane preparations were made as described by Unkles et al (2004a), with modifications. Mutants for blots were grown from a single point inoculum on one to two Petri dishes until the growth covered the plates. All of this growth was used to inoculate 400 ml MM with 10 mM proline and vitamins in a 1 L flask. This culture was grown for approximately 5 h 20 min (or longer if necessary to obtain the ideal growth phase) in an orbital shaker at 37 ˚C at 200 rpm. After this period 10 mM NaNO3 (10 mM NaNO2 with 1x p-amino benzoic acid supplement) was added to induce the cells over a 100 min (3 h) period. The mycelia were harvested through sterile Miracloth and washed with cold sterile water, then pressed in paper towels to dry before being frozen in liquid nitrogen in individual 300 mg pouches.

Mycelia (300 mg) were ground to a fine powder in liquid nitrogen and added to 10 ml of cold extraction buffer (NrtA protein extraction buffer, 50 mM sodium phosphate, 300 mM NaCl, 10 % v/v glycerol, 200 mM PMSF, 1 mM benzamidine, pH 7 with protease inhibitor tablet (Roche), NitA protein extraction buffer 20 mM Tris pH 7.5 (at 4 ˚C), 100 mM 6-amino hexanoic acid, 5 % (v/v) glycerol, 1 mM benzamidine). This was centrifuged at 3000 x g for 15 min at 4 ˚C in a Sorval SS-34 rotor (Sorval RC-6 plus). The supernatant was transferred to a fresh cold centrifuge tube and was centrifuged at 37,000 x g for 35 min at 4 ˚C. The supernatant was discarded and the pellet re-suspended in 80 µl cold extraction buffer. This extract was stored at -80 ˚C until ready for Western blotting.

SDS and blue native PAGE

Aliquots of each protein sample (50 µg) were run 10 % SDS-PAGE gels (see Appendix Five) at 200 V with 1 x sample buffer (60 mM Tris pH 6.8, 25 % v/v glycerol, 5 % w/v SDS, 1 % v/v saturated bromophenol blue) using 1x PAGE running buffer (250 mM Tris pH 8.3, 500 mM glycine, 1 % w/v SDS) with pre-stained protein size markers (New England Biolabs). This gel was then stained with Simply Blue Safe Stain (Invitrogen), following the manufacturer’s instructions to ascertain protein load. Once determined, a standardised aliquot of protein (50 µg) was loaded onto a gel for Western blot. Proteins were quantified with more accuracy using the Pierce Protein Quantification BCA kit following the manufacturer’s

instructions whereby 50 µg protein aliquots were used as standard for all Western analyses. For Western blotting of NrtA mutants, 10 % SDS-PAGE gels were run as standard. Gels were run with a low range protein size marker (Bio-Rad, Hertfordshire, U.K.) and each sample set was run in tandem with a positive and a negative control.

Sambrook et al (1989) discuss fully the theory and methods involved with SDS-PAGE. In SDS-PAGE, SDS is a dissociating agent which is used to coat the protein in charge. SDS denatures the protein’s tertiary structure and coats it in negative charge which is in proportion to mass; therefore allows proteins of differing charge to mass ratio run according to their size. Poly acrylamide gel is a synthetic gel which has variable pore size depending upon the concentration of acrylamide and crosslinker (bisacrylamide) used. PAGE is a discontinuous system whereby two gels of different buffered pH are employed to create an ion gradient, the first is known as the stacking gel, and the second, the separating or resolving gel. The function of the stacking gel and the ion gradient is to concentrate the proteins in a thin, sharp band. This is achieved by the preparation of the stack two pH units lower than the separating gel and electrophoresis buffer, also, the large pore size in the stack and small pore size in the separating gel. The running buffer used in PAGE contains glycine which is contains a source of trailing ion (glycinate) which runs behind the slowest of the proteins in the pH range.

For NitA mutants it was necessary to determine their quaternary structure using Blue

Native-Polyacrylamide Gel Electrophoresis (BN-PAGE). BN-PAGE refers to an

electrophoresis performed using Coomassie blue in cathode buffer. Native gels

employ non-denaturing conditions unlike SDS-PAGE gels; therefore separation is

based on the protein’s native charge to mass ratio. The methodology for BN-PAGE

was originally discussed in Schägger and van Jagow (1991) and is further discussed in

Eubel et al (2005).

In BN-PAGE experiments here the detergent dodecyl maltoside (DDM) was used to

lyse lipid membranes, not to provide charge unlike SDS in SDS-PAGE systems. The

other noteworthy difference with BN-PAGE is that the running buffer contains

Coomassie blue dye which is used to charge the protein for electrophoretic separation

(Eubel et al., 2005). In this method the proteins remain in their native state as regards

their secondary structure.

The gels for BN-PAGE were prepared using a multi-gel caster with a gradient former (SG Series, Hoeffer Scientific, Holiston, MA, U.S.A.) and a peristaltic pump. The 17 % gel was

loaded into the reservoir furthest from the pump and the 5 % added to the closest reservoir, with the stirrer bar active (in 5 % reservoir) the gel was allowed to mix as it was pumped into the multi-gel caster to create a linear gradient. Gels were overlaid with water-saturated iso- butanol to allow polymerisation. BN-PAGE was performed with a different buffering system to that of SDS-PAGE.

Standards and electrophoresis conditions

As there are no internal standards that can be employed for membrane proteins - particularly when studying the quaternary structure - standard proteins are employed as size standards. Proteins used here were maintained at concentrations of 10 mg ml-1_{, 1 µl of each protein was} loaded into one well on a gel with sample buffer (100 mM BisTris (Fluka, Hannover, Germany), 500 mM 6-amino hexanoic acid (Fluka), 5 % w/v Coomassie Blue G250 (Serva, Oxon, U.K.) pH 7) and extraction buffer to run alongside proteins in analysis. The standard proteins were Ferritin from equine spleen (440 kDa), Bovine serum albumin (66 kDa and 132 kDa) and Thyroglobulin from porcine thyroid (669 kDa). Protein extracts were incubated for one hour prior to electrophoresis with 1 % w/v DDM. Once the sample buffer was added they were then loaded into the 4 % stacking gel and run halfway into the separating gel at 100 V using Blue Cathode buffer (15 mM BisTris (Fluka) 50 mM Tricine (Fluka), 0.02 % w/v Coomassie Blue G250 (Serva), pH 7 at 4 ˚C) and separate anode buffer (50 mM BisTris (Fluka), pH 7 at 4 ˚C). At this point both the anode and cathode buffers were exchanged for fresh anode and cathode buffers (minus Coomassie) and the gel was then run to the end at 130 V. Once the run was complete, the gel was equilibrated in transfer buffer (25 mM Tris pH 8.3, 192 mM glycine, 20 % v/v methanol, 0.1 % w/v SDS) prior to Western transfer.

Western Transfer

Once protein gels had run they were prepared for Western transfer. Prior to transfer, gels were equilibrated in transfer buffer for 20 min. Hybond-P membranes were used for blotting and the apparatus for transfer was assembled using 3 MM paper and blotting pads. Hybond-P membrane was activated using methanol and equilibrated in transfer buffer. The transfer was performed on ice using a BioRad Mini Protean II system at 100 V for 50 min.

BN-PAGE membranes were washed in PAGE destain (45 % v/v ethanol, 10 % v/v acetic acid) for 10 min prior to washing in TBST (0.9 % w/v NaCl, 10 mM Tris pH 7.4, 0.1 % v/v Tween 20) to remove residual Coomassie blue which has a tendency to quench the reaction from the detection reagents. Membranes were washed for two intervals of 15 min in TBST at room temperature, and then incubated in 5 % block (GE Healthcare membrane blocking agent) in TBS (0.9 % NaCl, 10 mM Tris pH 7.4), overnight at 4 ˚C. After this incubation the

membranes were washed further in TBST for two periods of 10 min at room temperature. The membranes were then exposed to anti-V5HRP antibody (anti-V5HRP 1/5000 in TBS containing 0.5 % block) for at least 4 hr at room temperature). The probed membranes were then washed in TBST for two periods of 5 min and two periods of 20 min. TBST was drained from the membrane, placed protein side up, and was immersed in ECL plus detection solution (GE Healthcare Buckinghamshire, U.K.). After a 5 min incubation the detection solution was drained off and the membrane was placed in plastic bagging in an autoradiography cassette. A piece of Hyper-film ECL was applied to the gel, initially for a 30 second period though this was altered depending upon the resolution of bands on Hyperfilm ECL.

For this immunological detection an engineered sequence encoding the V5 epitope was fused to the 3’ end of the NrtA (and NitA) coding regions (Unkles, unpublished) (refer to Figure 2.1), using an anti-V5 antibody (Invitrogen) proteins on nylon membranes could be immunologically detected when the antibody was used in tandem with a Western blotting detection kit.