Positive-selection vector for direct protein expression.

We describe the development of a novel positive-selection vector, RHP-AmpS, that is suitable for seamless cloning and high-level protein expression in Escherichia coli. In this vector, beta-lactamase (Bla) was rendered nonfunctional by replacing the codon for the C-terminal amino acid of the beta-lactamase gene (bla) with a stop codon. Insertion of a target gene in the correct orientation (tail to tail) results in the reconstruction of the C-terminal codon (W290) of bla. This restores the function of the gene and allows the selection of positive recombinants on agar plates containing ampicillin. To allow a high level of protein expression, this selection cassette was inserted into the T7 polymerase-based expression cassette of the Novagen pET28a expression vector. To our knowledge, this is the first example of true positive-selection cloning and direct, high-level expression from a single vector.