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 strains. The affinity expression cassette polymerase chain reaction (AEC-PCR) allows the insertion of virtually any coding sequence in suitable expression vectors. For ease of purification of the (over)produced protein the gene expression cassettes are engineered by specifically designed oligonucleotide primers in the polymerase chain reaction (PCR) to contain 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 chromatography. In combination with PCR-mediated site-specific mutagenesis this 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 example, a string of six histidines has been engineered to either the N-terminal 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 produced in sufficient quantities to facilitate further studies on its immunological and molecular properties.
Raupach
B
B
Zentrum für Molekulare Biologie der Universität Heidelberg, ZMBH, FRG.
Marzillier
J
J
Schmidt
M A
MA
D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006639:Histidine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics; D037342:Pertussis Toxin; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D010566:Virulence Factors, Bordetella / Q000235:genetics*
D010455:Peptides; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; C033223:polyhistidine; D006639:Histidine; D011065:Poly(ADP-ribose) Polymerases; D037342:Pertussis Toxin