Research Project
3498005
Gene fusions have been constructed that promote the efficient secretion of human serum albumin (HSA) from Bacillus subtilis. HSA-encoding sequences are fused to promoter and secretion signal-encoding sequences derived from B. amyloliquefaciens alpha-amylase (amy) and neutral protease (npr) genes. However, secretion is efficient only when the level of synthesis of HSA is low. At higher synthesis levels, the protein remains cell- associated, with signal sequence attached. A procedure is proposed whereby B. subtilis mutants can be selected for improved ability to secrete high levles of HSA. The selection procedure makes use of tripartite gene fusions of the type amy-has-phoA and npr-hsa-phoA, in which sequences encoding E. coli alkaline phosphatase (phoA) are fused behind HSA-encoding sequences in the amy- and npr-has fusions. B. subtilis BR151 cannot grow on glycerol phosphate as sole carbon source, but it can grow if it contains a plasmid carrying an amy-phoA fused gene in which sequences encoding mature alkaline phosphatase are fused to the amy promoter and signal sequence. Strains containing the tripartite fusions do not grow on glycerol phosphate. Mutant derivatives of these strains able to grow on glycerol phosphate will be selected and characterized. Some are expected to show improved ability to secrete high levels of HSA.
