Bovine pancreatic phospholipase A2 (bPLA2) is a 14 kDa (123 amino acids) enzyme with seven disulphide bonds. Its primary function in vivo is the hydrolysis of dietary phospholipids. A T7 RNA polymerase based expression system was used to overexpress a synthetic gene encoding bovine pancreatic pro-phospholipase A2 in E. coli. The expressed protein was directed to the periplasmic space via an outer membrane secretory signal (OmpT) where, after translocation the protein formed insoluble inclusion bodies. Translocation efficiency was significantly increased (from about 25% to over 90%) when protein expression was induced after the cells had reached the stationary phase of growth (O.D.600~2.8). Optimal conditions for refolding of bPLA2 were found to be by rapid dilution under anaerobic conditions, in the presence of 2 mM oxidised glutathione, 4 mM reduced glutathione, 5 mM potassium EDTA and 25 mM sodium borate pH 8.7, with a final protein concentration of approximately 45 mg/L. The final amount of active recombinant protein produced was 22 mg per litre of bacterial culture. Site directed mutagenesis was used to investigate the role of the 58-71 surface loop of bovine pancreatic PLA2 in interfacial binding to a variety of aggregated phospholipids and surfactants. The surface loop was mutated so that the amino acid sequence was similar to that found in porcine pancreatic PLA2. Three mutants were made,Val-63Phe-63 (V63F), Asn-71Glu-71 (N71E) and finally the double mutant (V63FN71E). The mutants were overexpressed in E. coli and refolded in vitro. V63F had an increased affinity for lipid-water interfaces so that its binding to zwitterionic micelles was more like that of the porcine enzyme but catalysis was still similar to the bovine enzyme. Both N71E and V63FN71E showed a reduction in binding to zwitterionic micellar interfaces at pH 8 compared with bPLA2. The affinity for the lipid-water interface could be restored by the addition of a negative charge (via an anionic detergent) to the micellar interface.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:674494 |
| Date | January 1996 |
| Creators | Basran, Amrik |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/35251 |
Page generated in 0.01 seconds