Generation of monoclonal antibody against protein phosphatase 5

Aim. Serine/threonine protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatase family, comprises a regulatory tetratricopeptide repeat (TPR) domain and regulates many cell signaling pathways. Here, we describe the development of a PP5 specific monoclonal antibody (mAb) and characterize its suitability for Western blotting and immunoprecipitation. Methods. Hybridoma technology has been used for monoclonal antibody generation. Immunization was carried out with recombinant mouse PP5 expressed in Escherichia coli as a GST-tagged fusion protein. Results. mAb against PP5 has been generated. Conclusions. Generated mAb specifically recognizes recombinant and endogenous mouse and rat PP5 and is suitable for Western blotting and immunoprecipitation. This mAb will be useful tool for investigations of PP5 physiological role.

Recently we have identified PP5 as a new binding partner for a TSC2, a component of tuberous sclerosis complex TSC1/2 [28][29][30].TSC1/2 complex serves as a point of integration between growth-stimulatory and growth-suppressive signaling upstream of mTOR in PI3Ê/Akt/mTOR signaling pathway.The last one is implicated in signal transduction from mitogenic growth factors, nutrients, cellular energy levels and stress conditions to stimulate protein synthesis and cell growth [31,32].So far, very little is known about the molecular mechanisms of TSC2 dephosphorylation.Therefore, we hypothesized that the inter action with PP5 may control the phosphorylation status of TSC2.
To analyze the specificity of interactions between TSC2 and PP5 we developed monoclonal antibody (mAb), which recognizes PP5.Recombinant GSTtagged PP5 protein (GST-PP5) was used for mice immunization and hybridoma screening procedure.Here, we report that developed mAb specifically recognizes recombinant and endogenous PP5 in Western blotting and immunoprecipitation assay.This mAb could be a useful tool in study of PP5-mediated signal transduction pathways.
Materials and methods.Purification of GST-PP5 fusion protein.The bacterial expression system has been used for production of mouse full length GST-PP5 fusion protein.Generated plasmid pET42a/PP5 was transformed into Escherichia coli BL21 (lysE) and the expression of GST-PP5 fused protein was induced with 1 mM isopropyl-b-D(2)-thiogalactopyranoside (IPTG) for 3 h at 28 °C.Affinity purification of GST-PP5 was carried out under native conditions using Glutathione-Sepharose («Amersham», UK) as recommended by manufactures.The quality of the purified recombinant protein was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Production of hybridomas.Briefly, generation of mAb has been performed as described earlier [34].Female BALB/c mice (6-8 weeks old) were immunized with 20 µg of recombinant GST-PP5 fusion protein in complete Freund's adjuvant by intraperitoneal injections (i.p.) every two weeks.When the titer of anti-PP5 antibody in the serum of immunized mouse reached 10 -5 , the hybridomas production was performed according to a standard protocol [33].Immunized mice were boosted with 20 µg of antigen in phosphate-buffered saline (PBS) by i. p. injection.Three days later, spleenocytes from immunized mouse and SP2/0 myeloma cells were fused in the presence of PEG (MW 2000, «Merck», Germany).SP2/0 myeloma cells were cultured in RPMI 1640 medium containing 20 % fetal calf serum (FCS).Primary screening of hybridoma supernatants was performed 7 days later using ELISA.Isolated positive clones were subcloned by limiting dilution method [33].
ELISA assay.Polystyrene 96-well plates were loaded with GST-PP5 in the concentration 0.3 µg/well for 2 h at 37 °C.The plates were washed three times with PBS containing 0.1 % Tween-20 («Sigma», USA).For blocking of non-specific binding plates were incubated with 2 % bovine serum albumin (BSA) in PBS (pH 7.4) overnight at 4 °C (200 µl/well).Subsequently, the plates were loaded with 100 µl/well aliquots of hybri doma supernatant and incubated for 1 h at 37 °C, serum from immunized mouse was used as positive control.After three times washing, 100 µl of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibodies (1:5000 v/v, «Promega», USA) were added to each well following the incubation at 37 °C for 1 h.Plates were washed three times and substrate solution (0.02 % H 2 O 2 , 0.5 mg/ml 2.2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) sodium salt («Sigma») in 0.1 M citrate-phosphate buffer (pH 5.8)) was added to each well.After 15-min incubation at 37 °C, the absorbance of the each well was determined at 490 nm in an ELISA reader.
Hybridoma selection in Western blot analysis.Bacterially expressed GST-PP5 recombinant protein was boiled in sample buffer, resolved by a 10 % SDS-PAGE and electrotransferred to Immobilon-P membrane («Millipore», USA).The membrane was divided into strips, blocked by 0.5 % gelatin in PBS for 1 h at room temperature (RT) followed by a single wash with PBS containing 0.1 % Tween-20.Strips were incubated with either PBS, post-immune serum (1:1000), hybridoma supernatants from clones, or cell culture media alone for 4 h at RT.After three times washing, a HRPconjugated goat anti-mouse lgG («Promega») was added to the strips and incubated for 1 h at RT. Strips were washed three times, and developed by ECL Western blotting reagent («Amersham», Sweden).
Purification of mAb.For monoclonal antibody ascites fluid production BALB/c mice were injected with 0.5 ml of pristane and 7-10 days later inoculated with 5×10 6 of hybridoma cells [34].The ascitic fluid was collected after 7-10 days.The fraction of immunoglobulins was precipitated from ascitic fluid with 50 % ammonium sulphate, dialyzed overnight against PBS, pH 7.4 and purified by affinity chromatography using Protein A-Sepharose CL-4B («Amersham», Sweden).The IgG fractions were pulled together and dialyzed against PBS.The aliquots of purified antibodies were stored at -70 °C.
Production of polyclonal serum.Polyclonal serum has been collected from an immunized mouse by tail bleed before the hybridomas production was performed.Collected blood was incubated at 37 °C for 1 h then transferred to 4 °C for 2 h, and after that spinned down at 1500 rpm for 15 min at 4 °C.Serum was removed from cell pellet avoiding the packed one, diluted with equal volume of glycerol and stored at -20 °C.
Immunoprecipitation. Hybridoma supernatants from selected clones were incubated with 25 µl of 50 % suspension of Protein A-Sepharose CL-4B («Amersham», Sweden) for 2 h at 4 °C.Then, beads were washed three times in lysis buffer above and the lysates (500 µg of protein) from cell lines TSC2 +/+ p53 -/-MEFs cells, HEK 293, HeLa, CHO, MCF7, rat brain extract were added.After incubation o/n at 4 °C, beads were washed four times with 1 ml of lysis buffer.Immune complexes we-re removed from beads by boiling in Laemmli sample buffer and separated by SDS-PAGE.Resolved proteins were visualized by Coomassie staining or transferred onto PVDF membrane for immunoblotting.
Results and discussion.Recent progress toward understanding the mechanism of control on cell growth indicates that the tuberous sclerosis complex TSC1/2 as tumor suppressor an upstream negative regulator of mTOR activity.However, the regulation of tumor suppression by TSC1 and TSC2 is not well understood.We were interested in the elucidation of molecular mechanisms, which regulated TSC1/2 function in cellular response to various extracellular stimuli.In the previous study, by the yeast two-hybrid screening we identified already known and novel binding partners for TSC1 and TSC2 including Ser/Thr protein phosphatase 5 designated PP5 [28].To analyze the specificity of interactions between TSC2 and PP5 and to explore a potential effect of PP5 on TSC2 phosphorylation status we developed monoclonal antibody, which would specifically recognize PP5 in vivo.
Production and characterization of recombinant PP5 protein.We used the bacterial expression system to produce recombinant PP5 for mouse immunization and hybridoma clone screening.The full length of mouse PP5 was amplified by PCR and cloned into pET42a vector in frame with the GST-tag sequence to facilitate purification of recombinant protein.GST-PP5 was highly expressed upon IPTG induction and shown to be soluble under native conditions (Fig. 1, A).For GST-PP5 fusion protein purification Glutathione-Sepharose affinity chromatography was used (Fig. 1, B).
Hybridoma production and isolation of positive clones.BALB/c mice were immunized with 20 µg of purified GST-PP5 fusion protein in complete Freund's adjuvant every two weeks.Animals developed strong immune response to the antigen, and the titer of specific antibody reached 10 -5 in all immune sera after the third immunization.Immune spleen cells were fused with the myeloma cells Sp2/0 using PEG.After fusion cells were incubated in RPMI medium containing antibiotics, 20 % fetal bovine serum, and hypoxanthine/ aminopterin/thymidine (HAT) as a selective agent.Ten days later the first ELISA screening was carried out using GST-PP5 fusion protein to coat ELISA plate.We selected 14 positive hybridoma clones in the first round of screening.For selected clones a second ELISA screening was performed three days later and confirmed the GST-PP5 specificity for 4 clones.Selected clones were further subcloned twice using limiting dilution method.
Testing the specificity of selected clones in Western blotting.Supernatants from four positive clones were then tested by Western blotting using GST-PP5 protein.RPMI medium with 10 % FBS was used as a negative control.The serum collected from GST-PP5 immunized mouse was used as positive control.The obtained results clearly indicate that only one mAB, which designated as A4/1, recognizes a protein at the level corresponding to GST-PP5 (Fig. 2).
Produced antibodies recognize endogenous PP5.To determine whether the selected A4/1 mAb can recognize endogenous PP5, we tested their specificity in Western blotting and immunoprecipitation assay using cell lysates from different cell lines and organ extracts from mouse and rat.For these purposes 50 µg of cell lysates from TSC2 +/+ p53 -/-MEFs, TSC2 -/-p53 -/-MEFs, HEK 293, HeLa cell lines and organ extracts from mouse and rat brain, kidney, liver, spleen, and lung were analyzed in Western blot with A4/1 mAb.We found that mAb A4/1 recognizes a protein of approximately 58 kDa in TSC2 +/+ p53 -/-MEFs and TSC2 -/-p53 -/-MEFs but not in HEK293 cell lines (Fig. 3, A).Moreover, we also found that A4/1 mAb recognizes specifically endogenous PP5 in immunoblot analysis of mouse and rat protein extracts from brain, kidney, liver, spleen and lung (Fig. 3, B).
Taken together, this study describes the production of monoclonal antibody, which recognizes mouse and rat PP5.Generated mAb could be suitable for various immunoassays, including ELISA, Western blotting and immunoprecipitation.This mAb could be a useful tool in study of PP5-mediated signaling under different condition.
Acknowledgements.This work was particularly supported by state Fund for fundamental researches, Grant N F46/457-2011 and the President of Ukraine for young scientists, project N GP/F36/095 on «Investigation of molecular mechanisms of regulation of TSC2 tumor supressor activity».

Fig. 1 .Fig. 2 .
Fig. 1.Analysis of expression and affinity purification of recombinant GST-PP5 by SDS-PAGE: A -expression of GST-PP5 in BL21 pLysE cells was induced by IPTG (harvested cells were lysed in lysis buffer and centrifuged at 13 000 rpm for 20 min; total cell lysates and the supernatants from the IPTG induced and non-induced cells were separated by SDS-PAGE and Coomassie stained); B -affinity purification of GST-PP5 protein on GST-sepharose under native conditions (the GST-PP5 expressing cells were incubated with the GST-sepharose as described in Material and methods; after extensive washing, bound proteins were eluted in buffer containing 250 mM imidazole; obtained fractions were resolved by SDS-PAGE and Coomassie stained); lane 1 -total lysate; lane 2 -lysate before GST-seph.binding; lane 3 -lysate after GST-seph.binding; lane 4 -BSA; lane 5 -Protein Marker; lane 6-9 -elution 1-4, respectively; lane 10 -blank; lane 11 -GST-sepharose after elution.The position of recombinant GST-PP5 is indicated by arrows