Table 2. Growth of E. coli Harboring Different Cloning Vectors
We therefore determined that the construct containing the bla promoter from pET17b was giving us a suitable window for the selection of positive recombinants. We reasoned that a concentration of 300 μg/ml was high enough to ensure that no false positive colonies would be able to grow. Using these selective conditions we performed a number of cloning reactions using either gfp (25,26) or mCherry (27) as target genes. Both analytical PCR and sequencing of the PCR products showed that vectors contained the target genes in their correct orientation, reconstructing W290 of the bla gene. In our current experience (more than 20 cloning experiments, 4–16 colonies per experiment sequenced), we have not seen any false-positive colonies. However, the appearance of false-positive colonies cannot be ruled out completely; cloning of aberrant PCR products that reconstruct W290 of the β-lactamase are theoretically possible, as are plasmid rearrangements. However, these events are occurring at a very low frequency and should therefore not interfere with the intended applications.
Since we set out to construct a positive-selection vector that is also suitable for direct and high-level protein expression, we assessed the vector expression capacities by expressing the two fluorescent proteins mCherry and GFP. Because the ampicillin-positive–selection cassette was located inside the protein expression cassette, we also had to investigate whether it would have any negative influence on the expression levels. As a reference, we also cloned the two genes into the original RHP-CcdB vector, which is missing the ampicillin selection cassette but is otherwise identical (Figure 2). We did not observe any negative effects of the ampicillin selection cassette on cell growth compared with the control vector RHP-CcdB (data not shown). The presence of ampicillin during expression did not affect cell growth or expression levels.
The mCherry construct was expressed at similar levels as the reference (~250 μg/ml). However, the GFP construct was expressed significantly higher in the positive selection vector than in the control (50 μg/ml and 6 μg/ml , respectively). This could be due to the longer mRNA sequence transcribed in these constructs. As the selection cassette was inserted in opposite transcription direction between the T7 promoter and T7 terminator, the reverse complementary sequence for bla was also transcribed by the T7 polymerase. This additional mRNA could protect the actual gene of interest from degradation by 3'-RNases.
The mCherry positive selection construct was grown also in a 1-L fermenter in an in-house modified, rich auto-induction medium. We were able to purify an amount of 1.4 g of the rubredoxin-tagged mCherry protein.
In this study, we have used homologous recombination (17) and type IIs restriction enzymes (16) for cloning inserts into the positive selection vector. Unfortunately the C-terminal end of the truncated bla does not allow using classic restriction enzyme cloning for the target gene insertion. However, other cloning methods such as ligation-independent cloning (28) are well suited for this.
We set out to construct a vector for positive-selection and direct, high-level protein expression. We have constructed a vector, RHP-AmpS, that provides a simple way to achieve highly efficient selection of positive recombinants (Figures 1 and 2). Moreover, this vector was shown to be suitable for the expression of high levels of protein. To our knowledge, there is currently no vector available combining these features as efficiently as this one does.
Address correspondence to Christian Ostermeier, Novartis Pharma AG, Forum 1, Novartis Campus, CH-4056 Basel, Switzerland. email: [email protected]
We thank Roger M. Benoit, Anke Blechschmidt, Daniela Scherer-Becker, and Ramon N. Wilhelm for their helpful advice and inspiring discussions. We also thank Armand Wanner and Pascale Rieder for carrying out all the DNA sequencing reactions. We are also grateful to David J. Allcock for reviewing this manuscript.
The authors declare no competing interests.
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