In one of the method, membrane proteins were prepared from CEM-SS cells by using Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce)

In one of the method, membrane proteins were prepared from CEM-SS cells by using Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Pierce). was used to detect the binding protein for Bt18 parasporal protein in CEM-SS cells and the identified protein was sent for N-terminal sequencing. NCBI protein BLAST was used to analyse the protein sequence. Double immunofluorescence staining techniques was applied to localise Bt18 and binding protein on CEM-SS cell. == Results == Anion exchange separation of Bt18 parasporal protein yielded a 68-kDa parasporal protein with specific cytotoxic activity. Polyclonal IgG (anti-Bt18) for the 68-kDa parasporal protein was successfully raised and purified. Receptor binding assay showed that Bt18 parasporal protein bound to a 36-kDa protein from the CEM-SS cells lysate. N-terminal amino acid sequence of the 36-kDa protein was GKVKVGVNGFGRIGG. NCBI protein BLAST revealed that the binding protein was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Double immunofluorescence staining showed co-localisation of Bt18 and GAPDH on the plasma membrane of the CEM-SS cells. == Conclusions == GAPDH has been well known as a glycolytic enzyme, but recently GAPDH was discovered to have roles in apoptosis and carcinogenesis. Pre-incubation of anti-GAPDH antibody with CEM-SS cells decreases binding of Bt18 to the susceptible cells. Based on a qualitative analysis of the immunoblot and immunofluorescence results, GAPDH was identified as a binding protein on the plasma membrane of CEM-SS cells for Bt18 parasporal protein. == Background == Bacillus thuringiensis(Bt) was initially characterised as an insect pathogen, and its insecticidal activity was attributed largely to parasporal proteins. Recent studies, however, have reported that non-insecticidal Bt strains are more widely distributed than insecticidal ones [1]. This raises the question Isosakuranetin of whether non-insecticidal parasporal proteins have any biological activity which is as yet undiscovered. In a pioneering study, it was reported that selective human cancer cell-killing activity is associated with some non-insecticidal Bt isolates resulting in a new category of Bt parasporal protein called parasporin. Parasporins are defined as bacterial parasporal proteins that are capable of preferentially killing cancer Isosakuranetin cells [2,3]. Mizukiet al., (2000) obtained the first parasporin by expressing thecrygene encoding the Cry31Aa protein (also known as parasporin-1), which exhibits strong cytotoxicity against human leukemic T cells (MOLT-4), but did not exhibit insecticidal or hemolytic activities [4]. This was followed by the identification of three more proteins, Cry46Aa (parasporin-2), Cry41Aa (parasporin-3) and Isosakuranetin Cry45Aa (parasporin-4) also with selective cytotoxic activities against cancer cells [5-7]. Recently two more parasporin (PS5Aa1 and PS6Aa1) were added in the parasporin nomenclature [8]. Interestingly, a Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit cytotoxic activity preferentially for human leukaemic T cells (CEM-SS) but is non-cytotoxic to normal T cells or other cancer cell lines such as HeLa, MCF-7 and HT-29 [9]. It was reported that Bt18 parasporal protein is cytotoxic to CEM-SS as 84% cell death was observed at 0.5 g/mL (CD50value of 0.1224 0.0092 g/mL) [9]. Bt18 produces parasporal protein, which is also non-hemolytic to human or rat erythrocytes after trypsin activation, shows therapeutic and diagnostic potential with regards to leukaemia. This finding has triggered interest in elucidating the mode of action of Bt18 parasporal protein. Questions arise on how Bt18 parasporal protein specifically recognise leukaemic T cells. Insecticidal Bt parasporal proteins are known to bind receptors on the insect brush border membrane and it is suggested that these receptors play a role in the specificity of insecticidal activity [10,11]. We hypothesise that Bt18 cell killing activity is receptor mediated in that Bt18 parasporal protein binds specifically to a binding protein on the plasma membrane. To identify the binding protein, qualitative analysis were performed on Bt18 and CEM-SS cells using immunoblot and immunofluorescent staining. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as a binding protein for Bt18. Rabbit polyclonal to HYAL2 == Methods == == Bacterial strains and growth conditions == The Bt isolates used in this study were from Institute for Medical Research (IMR), Malaysia. Bt collections and the subtypes were determined using H antigen serotyping. Bt isolates used were Bt18,Bacillus thuringiensis2 (Bt2), andBacillus thuringiensissubspjegathesan(Btj). The Bt isolates were cultured in nutrient broth supplemented with CaCl2(0.01%), MgCl2(0.08%) and MnCl2(0.07%).