Conditions for toxin solubilisation and digestion used by the research group that first characterised PS-3 involved a 1 hour incubation at 37°C in 50 mM sodium carbonate 1 mM EDTA (10.5 pH), followed by a 90 minute treatment with proteinase K (10 µg/ml) in the presence of 10 mM DTT reducing agent at 37°C (Yamashita et al., 2005). Here a characterization of recombinant PS-3 was carried out in order to find the optimum conditions for protein solubilisation, digestion and purification.
Many attempts were made to purify PS-3. Mono S HR 5/5 (strong cation exchange) and Phenyl Sepharose 6 Fast Flow column (hydrophobic interaction chromatography) proved unfruitful (data not shown). Successful purification (but not separation of toxic fragments) was achieved with the gel filtration resin Sephacryl S- 200. In gel filtration molecules in solution are separated according to their sizes and shapes as they pass through a column packed with a chromatographic medium. Sephacryl is a hydrophilic composite gel made by covalently cross-linking allyl dextran with N,N'-methylene bisacrylamide, that allows for protein purification in the range from 5,000 to 250,000 Daltons for globular proteins (Biotech, 2010). Successful purification and separation of toxic fragments was achieved with a 1 ml Resource Q column (strong anion exchange, GE Healthcare Life Sciences) connected to an ÄKTA Purifier – FPLC System. In anion exchange chromatography, negatively charged molecules are attracted to a positively charged solid phase. Ionic interaction between
the oppositely charged sample molecule and the column resin retains the sample, which is later eluted by increasing salt concentration. The predicted pI of PS-3 protoxin calculated by Compute pI/Mw Expasy tool (Elisabeth Gasteiger, 2005) is 6.18 and only slightly changes when proteolytically cleaved toxin sequence is input. Close to neutral pI should make PS-3 suitable for both cation and anion exchange methods. However because PS-3 fragments were previously successfully separated using anion exchange (Yamashita et al., 2005) and because previous attempts to use cation exchange purification failed, anion exchange method was optimised to separate PS-3 fragments.
Cell based assays were used to measure viability of PS-3 treated cells in vitro. Metabolic activity and ATP levels were used as viability indicators. Trypsin activated Cry1Ca was tested as a negative control in these and other cell assays for two reasons. It shares a similar three domain Cry toxin fold with PS-3 while having activity against lepidopteran insects and no known human cytocidal activity. Secondly, Cry1Ca was expressed in the same acrystalliferous Bt strain as recombinant PS-3. Most of the cell assays were performed with susceptible HepG2 cells. Additionally, the effect of PS-3 was assessed on cellular viability of other cell lines not examined before, like Burkitt's lymphoma and lymphoblastoid cells.
Measurement of cell membrane integrity in compromised cells was achieved using two cytotoxicity cell assays. CellTox-Green assay, using a non-toxic small cyanine dye (~661 Da), allows for continuous assessment of membrane permeability for up to 72 hours in the same set of cells. The dye is excluded from viable cells, whereas in cells with compromised membranes it binds to the minor grove of the DNA producing a fluorescent signal proportional to the amount of cells with disrupted membranes.
Another membrane permeability assay, CytoTox-Glo measures a distinct protease activity associated with a cytotoxic marker, which is released from cells that have lost membrane integrity. The substrate cannot cross the intact membrane of live cells but in the presence of the released protease generates a stable luminescent signal. Scientists speculate that the cellular protease responsible for this reaction is consistent with tripeptidyl peptidase II (Niles et al., 2007), which is a relatively big 138 kDa protein. Osmotic swelling resulting from toxin induced membrane damage was recorded using a differential interference contrast (DIC) microscope.
Additionally, changes in membrane permeability were monitored in electrophysiology experiments using both artificial bilayers and biological membranes. Electrophysiology allows the measurement of electrical properties of a membrane. In a simplified model, the membrane represents a charge storing capacitor separating two current conducting solutions. In the presence of a pore, ions driven by the electrochemical gradient pass through the pore and current can be detected. The current is directly proportional to the voltage potential as described by Ohm’s law: I=V/R, where V is potential, I current and R resistance (Millikan et al., 1917). The planar lipid bilayer (PLB) technique has been widely used to study the properties of pores induced by Cry toxins (Lorence, 1995, Peyronnet et al., 2001, Rausell et al., 2004, Schwartz et al., 1993, Schwartz et al., 1997, Slatin et al., 1990a, Walters et al., 1993). The advantage of the PLB method is having a simplified system with total control over experimental conditions like lipid composition, pH and ionic concentration. For this study, the most significant is the fact that being receptor-free, the PLB method enabled evaluation of the innate pore forming ability of PS-3. Patch clamp recordings from cells exposed to Cry toxins have been previously documented (Schwartz et al.,
1991, Stumpff et al., 2007). However in contrast to PLBs, biological complexity of the cell membrane and the presence of endogenous channels are the main reasons why this technique has not been fully exploited in the studies of PFTs. Here, the whole cell and single channel patch clamp experiments were attempted and conductance levels compared with each other and with values obtained in PLB experiments.
PEGs are linear, generally non-toxic to cells polyethers, but they act like spherical molecules in aqueous solutions (Scherrer and Gerhardt, 1971). Pore diameter can be estimated from the smallest PEG that is excluded from the cells and prevents osmotic swelling (Sabirov et al., 1993). Using this approach pore size was estimated for various proteins, including Bt toxins: Cry1C (Peyronnet et al., 2002), PS-2 (Kitada et al., 2006). This method was also applied to estimate the size of PS-3 induced primary lesions.