A generic building block for C- and N-terminal protein-labeling and protein-immobilization.
Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.
Watzke
Anja
A
Max-Planck-Institut für molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Gutierrez-Rodriguez
Marta
M
Köhn
Maja
M
Wacker
Ron
R
Schroeder
Hendrik
H
Breinbauer
Rolf
R
Kuhlmann
Jürgen
J
Alexandrov
Kirill
K
Niemeyer
Christof M
CM
Goody
Roger S
RS
Waldmann
Herbert
H
D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D008301:Maleimides / Q000138:chemical synthesis / Q000737:chemistry*; D015394:Molecular Structure; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship; D018631:ras Proteins / Q000737:chemistry
D001386:Azides; D005456:Fluorescent Dyes; D008301:Maleimides; D011506:Proteins; D019204:GTP-Binding Proteins; D018631:ras Proteins