Gestión de Inventario

The Glutathione S-Transferase (GST) gene fusion system (Amersham Biosciences, GE Healthcare, Buckinghamshire, UK) enables the high level expression of native active gene fusions to the Schistosoma japonicum GST gene. GST fusions can then be purified from bacterial lysates using affinity chromatography by glutathione immobilised as glutathione sepharose. The GST gene fusion system was employed as a versatile system to detect protein-protein interactions between GST tagged proteins expressed in bacteria with mammalian cell lysates.

2.10.4.1 Expression of Recombinant GST Fusion Proteins in Bacteria

cDNA encoded within the pGEX 4T-1 plasmid (27-4580-01, Amersham Biosciences, GE Healthcare, Buckinghamshire, UK) was transformed into the chemically competent E.coli strain BL21(DE3), suitable for propagation and efficient protein expression (section 2.3.2), with ampicillin selection (100µg/ml). A single colony was picked and inoculated a 10ml LB/Ampicillin culture (100µg/ml Ampicillin) grown overnight at 37°c with shaking at 220 rpm. The culture was then scaled up into a 50ml LB/Ampicillin culture at a ratio of 50:1 and incubated for a further 3 hours at 37°c with shaking. The pGEX 4T-1 plasmid contains an isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible promoter to induce high level expression of the desired GST fusion protein. 200µm IPTG was administered to the culture, which was grown for a further 3 hours at 37°c with gentle shaking. The cells were then collected by centrifugation at 4000 rpm for 15 minutes. The supernatant was discarded and the cells freeze/thawed overnight at -30°c to aid lysis. The cells were then thoroughly re-suspended in 1ml of 1 x PBS. The cells were maintained on ice and lysed by sonication. Cells were exposed to 5 x 15

88 second bursts at the highest intensity, with 15 second intervals between each burst. The lysates were then centrifuged at 13,000 rpm for 10 minutes to pellet the insoluble cell debris. The supernatant was retained and stored at - 20°c for future use.

2.10.4.2 Purification of Recombinant GST Fusion Proteins from Bacterial Lysates

GST fusion proteins can be purified from bacterial lysates due to the high affinity of the GST moiety for glutathione. Glutathione immobilised on sepharose (Glutathione Sepharose™ 4B, 17-0756-01, Amersham Biosciences, GE Healthcare, Buckinghamshire, UK) was stored at 4°c preserved in a 20% (v/v) ethanol solution. Prior to use the Glutathione Sepharose™ 4B was washed twice with 1ml of 1 x PBS to remove the ethanol solution. The appropriate amount of the bacterial lysate expressing the GST fusion alone (as a control) and the GST-tagged protein of interest was added to 30µl of washed glutathione sepharose and incubated in a total volume of 1ml, made up by 1 x PBS, for 20 minutes with rotation at 4°c. The beads were collected by centrifugation at 2000rpm for 1 minute and the supernatant containing the bacterial lysate was discarded. In order to remove proteins non-specifically bound to the Glutathione Sepharose™ 4B, the beads were washed twice with 1ml of RIPA buffer for 5 minutes followed by collection by centrifugation at 2000rpm. The GST fusion proteins may then be eluted from the Glutathione Sepharose™ 4B beads in denaturing conditions by the addition of 30µl of 5x SDS-PAGE sample buffer. Samples were then analysed by SDS-PAGE (section 2.9.1) and acrylamide gels were then coomassie stained (section 2.9.5) to quantify the GST fusion protein expression and purification.

89 2.10.4.3 GST fusion – Protein Interaction Assays

GST fusions immobilised onto Glutathione Sepharose™ 4B were then subjected to prey interacting proteins from human cell lysates or proteins expressed in vitro in Rabbit Reticulocyte lysate. The GST fusion proteins immobilised onto Glutathione Sepharose™ 4B beads were then blocked by incubation in 1ml of 5% (w/v) BSA in 1 x PBS for 1 hour at 4°c with rotation. This blocking step minimalises the amount of non-specific binding of the prey protein to the GST fusion, to the beads or to the eppendorf tube. The beads were pelleted by centrifugation at 2000 rpm for 1 minute and the blocking solution removed. The beads were then incubated with the prey protein, either expressed in vitro (prepared as described in section 2.10.4.4) or as human cell lysate overnight at 4°c with rotation. For in vitro transcribed and translated [35S]-methionine labelled proteins, 1-20µl of the reaction mixture was added to the beads in 1ml of 5% (w/v) BSA in 1 x PBS. Human cell lysates were prepared as for immunoprecipitations (described in section 2.10.1), lysed in 500µl of cold RIPA, and incubated with the glutathione immobilised GST fusions in an additional 500µl of 10% (w/v) BSA in 1 x PBS, so that the final concentration of BSA is maintained at 5%. Prior to incubation of the prey protein to the beads, an appropriate input was aliquoted in order to evaluate the degree of protein interaction achieved by the assay.

Following overnight incubation, the beads were pelleted by centrifugation at 2000rpm for 1 minute and the supernatant discarded. The beads were washed 3 times for 5 minutes in 1ml of the incubation buffer (RIPA or PBS). The GST fusion proteins were eluted by re-suspension in 30µl of 5 x SDS sample buffer. The proteins were then resolved by SDS-PAGE and then stained using coomassie blue stain (section 2.9.5). Experiments utilising proteins expressed in vitro in rabbit reticulocytes, were analysed for protein interaction by drying of the polyacrylamide gel and then autoradiography to detect the [35S]-methionine label of the prey protein (section 2.10.4.5). For interaction assays using human cell lysates, the eluate was resolved by SDS-

90 PAGE and then analysed by immunoblot for the presence of the interacting protein.

2.10.4.4. In Vitro Transcription and Translation

To evaluate whether a protein-protein interaction is direct in its nature, rather than indirect via an additional protein, GST fusion proteins were subjected to proteins expressed in vitro in rabbit Reticulocyte lysate. As Rabbit reticulocytes have minimal cellular proteins except those required for globin production, an indication regarding the nature of the protein-protein interaction can be deduced. The TNT® Coupled Reticulocyte Lysate System (Promega, WI, USA) was utilised to generate radio-labelled prey protein. The components of the system were removed from storage at -80°c. The TNT®

T7 RNA polymerase was immediately placed on ice whilst the TNT®

Reticulocyte lysate was thawed by hand and the other components were thawed at room temperature before incubation on ice. The following components for a standard TNT® lysate reaction were then assembled in a 0.5ml eppendorf tube on ice:

Component Volume (µl)

TNT® Rabbit Reticulocyte Lysate 27.5

TNT®Reaction Buffer 2

TNT®T7 RNA Polymerase 1

Amino Acid Mixture, Minus Methionine, 1mM 1

[35S] methionine (Ci/mmol) 2

RNasin® Ribonuclease Inhibitor (40U/µl) 1

DNA template (0.5µg/µl) 2

HPLC grade H2O to a final volume of 50

The reaction mixture was then mixed by vortexing and then incubated at 30°c for 90 minutes. The translation product was then either immediately used (section 2.10.4.3) or stored at -30°c for future use.

91 2.10.4.5 Gel drying and Auto-Radiography

Coomassie Blue stained SDS-PAGE acrylamide gels were placed onto Whatman filter paper and overlaid with cling film. Gels were then dried using a BioRad Gel Dryer (model 583) for 2 hours. The cling film was then removed and the gels placed into a developing cassette. Under red light ECL Hyperfilm (Amersham™) was exposed to the gel and then incubated for 24- 72 hours at -80°c. The cassette was then allowed to thaw and the film was then hand developed.