한빛사논문
- 조회1,314
Abstract
Ivana Nikić1,2, Jun Hee Kang1,2, Gemma Estrada Girona1,2, Iker Valle Aramburu1 & Edward A Lemke1
1Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. 2These authors contributed equally to this work.
Correspondence should be addressed to E.A.L.
Abstract
We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron–demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced-by site-directed mutagenesis-at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4–7 d, followed by the described transfections and labeling reaction steps, which can take 3–4 d.
논문정보
- Research article
- 2015년 04월 (BRIC 등록일 2015-05-18)
- 국외 연구진
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