A T7 EXPRESSION VECTOR OPTIMIZED FOR SITE-DIRECTED MUTAGENESIS USING OLIGODEOXYRIBONUCLEOTIDE CASSETTES

Site-directed mutagenesis is widely used to examine structure/function relationships in proteins. We have designed a bacterial expression vector series which is optimized for efficient site-directed mutagenesis and subsequent protein synthesis without intervening subcloning steps. The vectors, derived from the T7 expression vectors of Studier and his collaborators [Studier et al., Methods Enzymol. 185 (1990) 60-891, are small and have a bacteriophage f1 origin of replication for production of single-stranded (ss) DNA. Both single-site mutants [using ssDNA and mutating oligodeoxyribonucleotides (oligos)] and cassette mutants (mutagenesis of a short region by inserting double-stranded oligos into unique restriction sites) are rapidly synthesized and expressed with these vectors. Vector construction and use are detailed with examples showing the expression of the sequences encoding human carbonic anhydrases II and III. Production levels of greater than 60 mg of protein per liter of culture have been obtained.