2, Novartis Institutes for BioMedical Research, Basel, Switzerland
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.
As more and more genome sequences become available, researchers are presented with a multitude of new, not-yet described proteins. For many experiments, these proteins have to be overexpressed and purified to have sufficient material for analysis. In addition to cloning the full-length gene into an expression vector, multiple, truncated, and/or mutated versions of the gene often have to be cloned and expressed to obtain sufficient and soluble protein for downstream applications. However, cloning reactions are very inefficient processes and vector background from parental plasmids (template vector and cloning vector) can make it very tedious to find the right clone. In modern high-throughput structural biology laboratories, an increasing number of gene constructs need to be cloned and expressed as quickly as possible in order to characterize their protein products and interactions in a timely manner. Reducing the time and effort spent on identifying the correct clones reduces costs significantly and leads to higher throughput at the cloning interface. Various ways to address the problem of parental vector background have been described (1). However, all of these classical methods still have a considerable background, need unusual selection media, or are not suitable for direct protein expression.
To facilitate the selection of correct recombinants, a number of positive-selection vectors have been developed; in these vectors, the successful cloning of DNA fragments results in an obvious change of phenotype. The positive selection in these vectors is achieved either by the inactivation of a genetic marker (2,3,4,5,6,7,8,9) or the replacement of said marker by the target gene (10,11,12,13). (For a detailed review on positive selection vectors, see Reference 14,.) These powerful selection strategies, however, are often only suitable for cloning; direct high-level protein expression from the cloning vector is not possible. In addition, most of the known selection systems have no active, positive selection toward the correct insert in its correct orientation and therefore incorrectly inserted fragments can result in a surviving phenotype.
Huang et al. (15) found that 43 out of the 263 amino acids that form the core protein of β-lactamase (Bla) are essential for its full function. Interestingly, the last amino acid at the C-terminus of Bla, tryptophan (W290), was one of the residues that did not tolerate any changes. We reasoned that using this observation, a positive-selection vector could be constructed containing a truncated version of bla that is missing the C-terminal codon for W290. Cloning a target gene carrying the respective sequence to reconstruct this amino acid residue at the C-terminus of Bla into the vector would result in restoring the protein's full functionality (Figure 1). Colonies carrying the correct insert in the correct orientation would therefore be ampicillin-resistant and could be easily selected. However, no fusion protein with Bla is created, as the two genes are oriented in opposing transcriptional directions and are each separated by two stop codons, respectively.Figure 1.
By using this approach, we have developed the vector RHP-AmpS (rubredoxin/ His6/PreScission ampicillin selection; Figure 2, GenBank accession no. FJ545754), which is capable of both positive selection and direct, high-level protein expression.Figure 2.
All PCR reactions were performed using Phusion High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in accordance with the manufacturer's protocol. Primers were designed to have a melting temperature (Tm) of ≥65°C using the Finnzymes Tm calculator (www.finnzymes.com/tm_determination_old.html). Primers used for cloning with homologous recombination had a 20-bp 5' non-annealing tail homologous to the termini of the linearized vector. For cloning into the positive selection vector RHP-AmpS, the reverse primer also contained the sequence to restore the C-terminal tryptophan of Bla and a double stop codon for selective marker and target gene, respectively.