Published online 8 July 2005
Methods Online |
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
Program in Cellular Biotechnology, Institute of Biotechnology Viikki Biocenter, PO Box 56, FI-00014 University of Helsinki Finland 1VTT Biotechnology PO Box 1500, FI-02044, VTT, Finland
*To whom correspondence should be addressed. Tel: +358 9 19159516; Fax: +358 9 19159366. Email: harri.savilahti{at}helsinki.fi
Received April 1, 2005. Revised June 10, 2005. Accepted June 10, 2005.
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.
Present address: Jussi Jäntti, Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, PO Box 56, FI-00014, University of Helsinki, Finland
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