Avoiding the ends: internal epitope tagging of proteins using transposon Tn7.
Peptide tags fused to proteins are used in a variety of applications, including as affinity tags for purification, epitope tags for immunodetection, or fluorescent protein tags for visualization. However, the peptide tags can disrupt the target protein function. When function is disrupted by fusing a peptide to either the N or C terminus of the protein of interest, identifying alternative ways to create functional tagged fusion proteins can be difficult. Here, we describe a method to introduce protein tags internal to the coding sequence of a target protein. The method employs in vitro Tn7-transposon mutagenesis of plasmids for random introduction of the tag, followed by subsequent Gateway cloning steps to isolate alleles with mutations in the coding sequence of the target gene. The Tn7-epitope cassette is designed such that essentially all of the transposon is removed through restriction enzyme digestion, leaving only the protein tag at diverse sites internal to the ORF. We describe the use of this system to generate a panel of internally epitope-tagged versions of the Saccharomyces cerevisiae GPI-linked membrane protein Dcw1 and the Candida glabrata transcriptional regulator Sir3. This internal protein tagging system is, in principle, adaptable to tag proteins in any organism for which Gateway-adapted expression vectors exist.
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.
Rebecca E
RE
Zordan
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.
Brian J
BJ
Beliveau
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.
Jonathan A
JA
Trow
Department of Molecular Biology and Genetics, The Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.
Nancy L
NL
Craig
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185 bcormack@jhmi.edu.
Brendan P
BP
Cormack
D001483:Base Sequence; D002175:Candida / Q000235:genetics; D004251:DNA Transposable Elements; D000939:Epitopes / Q000235:genetics*; D008361:Mannosidases / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
D004251:DNA Transposable Elements; D000939:Epitopes; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D008361:Mannosidases; C470737:DCW1 protein, S cerevisiae
Saccharomyces; epitope tag; protein tagging; transposition