Background: During standard gene cloning, the recombinant protein appearing in bacteria as the result of expression leakage very often inhibits cell proliferation leading to blocking of the cloning procedure. Although different approaches can reduce transgene basal expression, the recombinant proteins, which even in trace amounts inhibit bacterial growth, can completely prevent the cloning process.
Methods: Working to solve the problem of DNase II-like cDNA cloning, we developed a novel cloning approach. The method is based on separate cloning of the 5' and 3' fragments of target cDNA into a vector in such a way that the short Multiple Cloning Site insertion remaining between both fragments changes the reading frame and prevents translation of mRNA arising as a result of promoter leakage. Subsequently, to get the vector with full, uninterrupted Open Reading Frame, the Multiple Cloning Site insertion is removed by in vitro restriction/ligation reactions, utilizing the unique restriction site present in native cDNA.
Results: Using this designed method, we cloned a coding sequence of AcDNase II that is extremely toxic for bacteria cells. Then, we demonstrated the usefulness of the construct prepared in this way for overexpression of AcDNase II in eukaryotic cells.
Conclusions: The designed method allows cloning of toxic protein coding sequences that cannot be cloned by standard methods.
General significance: Cloning of cDNAs encoding toxic proteins is still a troublesome problem that hinders the progress of numerous studies. The method described here is a convenient solution to cloning problems that are common in research on toxic proteins.
Keywords: Acanthamoeba castellanii; Frameshift introduction; Nuclease genes; Protein overexpression; Toxic protein.
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