A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach.
Bacteriophage Mu in vitro transposition constitutes a versatile tool in molecular biology, with applications ranging from engineering of single genes or proteins to modification of genome segments or entire genomes. A new strategy was devised on the basis of Mu transposition that via a few manipulation steps simultaneously generates a nested set of gene constructions encoding deletion variants of proteins. C-terminal deletions are produced using a mini-Mu transposon that carries translation stop signals close to each transposon end. Similarly, N-terminal deletions are generated using a transposon with appropriate restriction sites, which allows deletion of the 5'-distal part of the gene. As a proof of principle, we produced a set of plasmid constructions encoding both C- and N-terminally truncated variants of yeast Mso1p and mapped its Sec1p-interacting region. The most important amino acids for the interaction in Mso1p are located between residues T46 and N78, with some weaker interactions possibly within the region E79-N105. This general-purpose gene truncation strategy is highly efficient and produces, in a single reaction series, a comprehensive repertoire of gene constructions encoding protein deletion variants, valuable in many types of functional studies. Importantly, the methodology is applicable to any protein-encoding gene cloned in an appropriate vector.
Poussu
Eini
E
Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, PO Box 56, FI-00014, University of Helsinki, Finland.
Jäntti
Jussi
J
Savilahti
Harri
H
D010583:Bacteriophage mu / Q000235:genetics*; D001665:Binding Sites; D004251:DNA Transposable Elements; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D004251:DNA Transposable Elements; C106953:MSO1 protein, S cerevisiae; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins