Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy.
A genetically modified form of the human DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase (hAGT) was used to immobilize different recombinant hAGT fusion proteins covalently and selectively on gold and glass surfaces. Fusion proteins of hAGT with Glutathione S-Transferase and with tandem repeats of Titin Ig-domains, were produced and anchored via amino-polyethylene glycol benzylguanine. Anchoring was characterized and quantified with surface plasmon resonance, atomic force microscope and fluorescence measurements. Individual fusion proteins were unfolded by single molecule force spectroscopy corroborating the selectivity of the covalent attachment.
Kufer
Stefan K
SK
Lehrstuhl für Angewandte Physik, Sektion Physik, Ludwig-Maximilians-Universität München and Center for NanoScience, Amalienstrasse 54, 80799 Munich, Germany.
Dietz
Hendrik
H
Albrecht
Christian
C
Blank
Kerstin
K
Kardinal
Angelika
A
Rief
Matthias
M
Gaub
Hermann E
HE
D001665:Binding Sites; D064211:Connectin; D004260:DNA Repair; D005898:Glass / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry; D018625:Microscopy, Atomic Force / Q000379:methods*; D009124:Muscle Proteins / Q000737:chemistry; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D020349:Surface Plasmon Resonance / Q000379:methods; D020080:Tandem Repeat Sequences; D013997:Time Factors
D064211:Connectin; D007136:Immunoglobulins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C577285:TTN protein, human; D006046:Gold; D019853:O(6)-Methylguanine-DNA Methyltransferase; D005982:Glutathione Transferase; D011494:Protein Kinases