Human anaphylatoxin C3a : assay and recombinant fusion protein expression in Saccharomyces cerevisiae
Native human C3a has been purified from plasma in an active form, setting up what is now a routine purification procedure in this laboratory. An assay system has been selected from a range of methods for measuring the activity of C3s. This method measures the release of histamine from human peripheral blood basophils in response to the interaction between C3a and the cell surface receptor. The quantitation of histamine release is based on the condensation of histamine with orthophthalaldehyde to form a fluorescent product with three stereoisomers, which can be separated and quantified using HPLC with fluorescence detection. The coding sequence for human anaphylatoxin C3a has been cloned into the PGK structural gene of the yeast/E. coli shuttle vector pMA27 to create the coding sequence for a PGK-C3a fusion protein. The DNA code for the recognition and target sequence for the proteolytic enzyme blood coagulation factor Xa (lle-Glu-Arg) was also introduced, using oligonucleotide-directed mutagenesis, between the PGK and C3a codes, to enable in vitro cleavage of the proteins to be carried out subsequent to expression in a suitable microbial host. The Saccharomyces cerevisiae host strain MT302/28b has been transformed with the plasmid encoding the fusion product and expression of the desired protein detected.