Abstract
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 |
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Keywords
- Mu in vitro
- transposition
- gene cloning
- gene expression
- genes
- Protein production
- proteins
Cite this
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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. / Poussu, Eini; Jäntti, Jussi; Savilahti, Harri.
In: Nucleic Acids Research, Vol. 33, No. 12, e104, 2005.Research output: Contribution to journal › Article › Scientific › peer-review
TY - JOUR
T1 - A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies
T2 - Mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach
AU - Poussu, Eini
AU - Jäntti, Jussi
AU - Savilahti, Harri
PY - 2005
Y1 - 2005
N2 - 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.
AB - 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.
KW - Mu in vitro
KW - transposition
KW - gene cloning
KW - gene expression
KW - genes
KW - Protein production
KW - proteins
U2 - 10.1093/nar/gni102
DO - 10.1093/nar/gni102
M3 - Article
VL - 33
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 12
M1 - e104
ER -