A novel method for cloning of coding sequences of highly toxic proteins

• Published in: Biochimica et biophysica acta. General subjects Vol. 1863; no. 3; pp. 521 - 527
• Main Authors: Krela, Rafal, Poreba, Elzbieta, Weglewska, Martyna, Skrzypczak, Tomasz, Lesniewicz, Krzysztof
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2019
• Subjects: Acanthamoeba castellanii; bacteria; bacterial growth; cell proliferation; complementary DNA; deoxyribonucleases; Frameshift introduction; gene overexpression; messenger RNA; molecular cloning; Nuclease genes; nucleotide sequences; Protein overexpression; recombinant proteins; Toxic protein; toxicity; transgenes; translation (genetics); Nuclease genes; Toxic protein; Acanthamoeba castellanii; Protein overexpression; Frameshift introduction
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2018.12.010

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. 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. 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. The designed method allows cloning of toxic protein coding sequences that cannot be cloned by standard methods. 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. [Display omitted] •A novel method for cloning of the toxic proteins cDNAs was developed.•Insertion introduced into cDNAs prevents the toxic effect of transgene leakage.•The sequences cloned in this way can be used to overexpress toxic proteins.