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.
Haag
Andreas F
AF
Department of Biotechnology & Bioinformatics, Weihenstephan University of Applied Sciences, Freising, Germany.
Ostermeier
Christian
C
D000668:Ampicillin Resistance / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D012641:Selection, Genetic
D011506:Proteins