Abstract
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.
Original language | English |
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Article number | e104 |
Number of pages | 8 |
Journal | Nucleic Acids Research |
Volume | 33 |
Issue number | 12 |
DOIs | |
Publication status | Published - 2005 |
MoE publication type | A1 Journal article-refereed |
Keywords
- Mu in vitro
- transposition
- gene cloning
- gene expression
- genes
- Protein production
- proteins