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

Nucleic Acids Res. 2005 Jul 8;33(12):e104. doi: 10.1093/nar/gni102.


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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriophage mu / genetics*
  • Binding Sites
  • DNA Transposable Elements*
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • Munc18 Proteins
  • Nerve Tissue Proteins / metabolism*
  • Plasmids
  • Protein Engineering / methods*
  • Protein Interaction Mapping*
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Deletion*
  • Vesicular Transport Proteins / metabolism*


  • DNA Transposable Elements
  • MSO1 protein, S cerevisiae
  • Membrane Proteins
  • Munc18 Proteins
  • Nerve Tissue Proteins
  • Saccharomyces cerevisiae Proteins
  • Vesicular Transport Proteins