EFFICIENT PRODUCTION OF ACTIVE AND MUTATED ADP-RIBOSYLTRANSFERASE (S1) OF PERTUSSIS TOXIN USING AFFINITY EXPRESSION CASSETTE POLYMERASE CHAIN-REACTION

We describe an efficient, general approach for cloning, expression and purification of heterologous proteins in Escherichia coli host strain s. The affinity expression cassette polymerase chain reaction (AEC-PCR ) allows the insertion of virtually any coding sequence in suitable ex pression vectors. For ease of purification of the (over)produced prote in the gene expression cassettes are engineered by specifically design ed oligonucleotide primers in the polymerase chain reaction (PCR) to c ontain either 3' or 5' additional nucleotides coding for a short amino acid sequence constituting an 'affinity block' fused to either end of the protein. This allows a one-step purification by affinity chromato graphy. In combination with PCR-mediated site-specific mutagenesis thi s approach is a highly efficient way for the expression and isolation of proteins and for the analysis and dissection of functional domains. The application of AEC-PCR is demonstrated by the cloning, production and purification of the native, active and the mutagenized, inactive ADP-ribosyltransferase (S1 subunit) of pertussis toxin. In this exampl e, a string of six histidines has been engineered to either the N-term inal or the C-terminal end of the protein to serve as 'affinity block' for the isolation of the recombinant protein by immobilized metal ion affinity chromatography (IMAC). Thus, the S1 subunit can now be produ ced in sufficient quantities to facilitate further studies on its immu nological and molecular properties.