FLAG-tagged bacterial alkaline phosphatase (FLAG-BAP) and unconjugated enhanced green fluorescent protein (eGFP; pEGFP-N1) constructs were obtained from manufacturers
FLAG-tagged bacterial alkaline phosphatase (FLAG-BAP) and unconjugated enhanced green fluorescent protein (eGFP; pEGFP-N1) constructs were obtained from manufacturers. Beverly, MA, USA). The resulting 3?kb bands were sub-cloned into either the TOPO-pCR-XL (Invitrogen) or pGEM T-easy vectors (Promega) and ligated into their final vectors. Single clones positive for full-length TPO were identified by restriction digest and in-frame integration of the cDNA confirmed by DNA sequencing. FLAG-tagged bacterial alkaline phosphatase (FLAG-BAP) and unconjugated enhanced green fluorescent protein (eGFP; pEGFP-N1) constructs were obtained from manufacturers. The unconjugated eGFP construct will be referred to as eGFP-nil. Co-immunoprecipitation CHO cells at 70% confluence were co-transfected with FLAG-TPO, eGFP-TPO (10?g per 10?cm2 plate), or their respective controls: FLAG-BAP, eGFP-nil using Fugene HD (Roche, UK). After AICAR phosphate 18?h, proteins were extracted as previously described. Input samples were retained, and the remainder pre-cleared for non-specific binding with protein-G sepharose beads for 1?h. Samples were then centrifuged at 1,000?g at 4C for 1?min, the N-terminally tagged TPO fusion proteins were immunoprecipitated overnight using their corresponding antibodies: Anti-FLAG M2 (Sigma), 0.5?g, or GFP(FL) and GFP(B2; Santa Cruz Antibodies, CA, USA). Immunoprecipitating beads were thoroughly washed with lysis buffer and subjected to SDS-polyacrylamide gel electrophoresis (PAGE; as above) alongside their corresponding pre-immunoprecipitated input samples. Immunoblotting was performed as before with the following exceptions: the presence of eGFP-TPO was detected with anti-GFP murine monoclonal or FLAG-TPO using anti-FLAG M2. Tagged-TPO ELISA Extracts from CHO cells co-transfected with FLAG-TPO, and either eGFP-TPO or eGFP-nil were prepared as above. Samples with equal total protein, assessed by micro-BCA, were incubated overnight in enzyme-linked-immunosorbent assay (ELISA) plates pre-coated with anti-FLAG M2 mouse antibody (100?ng/well) and blocked with 2% BSA in PBST. After thorough washing with PBST, GFP(FL) rabbit antibody was added for 1?h and detected with anti-rabbit AP-conjugate. Development of para-nitrophenylphosphate (pNPP) was measured (A405) after 1?h. Bacterial expression of Fab fragments AICAR phosphate Four cloned Fabs, TR1.9 and TR1.8; and WR1.7 and SP1.4, which recognised the human TPO B and A immunodominant epitopes, respectively, were obtained (kind gift of Drs. B Rapoport and S McLachlan, LA) [19]. Starter cultures of XL1-blue made up of each of the Fab expression constructs were used to inoculate Superbroth: 30?g/l tryptone, 20?g/l yeast extract, 10?g/l 3-(assessments, paired by the day of experiment. Results High-molecular weight TPO isoforms exist in CHO-TPO cells Immunoblotting of extracts from the stably transfected CHO-TPO cell line with the monoclonal anti-TPO antibody mAb47 revealed AICAR phosphate a discrete 110?kDa band and additional high-molecular weight (HMW) bands in the 220C240?kDa range. These Mdk HMW complexes were resistant to denaturation with conventional reducing brokers (Fig.?1). The 110?kDa isoform is consistent with the expected size of monomeric TPO. As MPO is known to function as a homodimer, we suspected that this HMW-TPO isoforms could also be homodimers. High-molecular weight TPO is usually a homodimer FLAG- and eGFP-tagged TPO were transiently co-expressed in CHO cells, and cell extracts were immunoprecipitated using antibody-conjugated protein-G sepharose beads. Co-transfections using FLAG-BAP and eGFP-nil, along with the tagged TPO constructs were used as controls. Cell extracts from cells co-transfected with FLAG- and eGFP-TPO revealed an eGFP reactive band following immunoprecipitation with anti-FLAG coated AICAR phosphate beads (Fig.?2). Similarly, immunoprecipitation of the same extracts with anti-GFP coated beads showed an immunoreactive band to anti-FLAG antibody. Co-transfected cells with either FLAG-BAP or eGFP-nil showed no non-specific binding (Fig.?2). Open in a separate windows Fig.?2 Differentially tagged recombinant human thyroid peroxidase (rhTPO) forms dimeric complexes in Chinese hamster ovary (CHO) cells. CHO cells were co-transfected with FLAG- and enhanced green fluorescent protein (eGFP)-TPO, or their respective control constructs FLAG-BAP or eGFP-nil. Cell extracts were immunoprecipitated via their fusion-tags using either anti-FLAG M2, anti-GFP B2, or anti-GFP FL antibody bound to protein-G sepharose. a FLAG-BAP (and and test) in binding of Graves or control sera at MMI concentrations up to 50?mM was observed. c Four Fabs, specific for the TPO IDR were studied in triplicate. Following incubation with MMI at concentrations of 500?nMC50?mM, there were no alterations in Fab binding to rhTPO. Substrate absorbance was dramatically reduced (p??0.01) at 500?mM MMI in all experiments (we believe a non-specific effect, possibly related to high ionic content; see Results). Data presented as means with bars representing SEM Effect of MMI on TPO localisation in CHO-TPO cells Membrane localisation has been proposed as the site of enzymatically active mature TPO. We wondered whether thionamide could affect TPO localisation. CHO-TPO cells were.