Positive-selection vector for direct protein expression

Abstract

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, β-lactamase (Bla) was rendered nonfunctional by replacing the codon for the C-terminal amino acid of the β-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.