Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy

Eur Biophys J. 2005 Dec;35(1):72-8. doi: 10.1007/s00249-005-0010-1. Epub 2005 Sep 14.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Connectin
  • DNA Repair
  • Glass / chemistry
  • Glutathione Transferase / metabolism
  • Gold / chemistry
  • Humans
  • Immunoglobulins / chemistry
  • Microscopy, Atomic Force / methods*
  • Muscle Proteins / chemistry
  • O(6)-Methylguanine-DNA Methyltransferase / genetics
  • O(6)-Methylguanine-DNA Methyltransferase / metabolism*
  • Protein Kinases / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Spectrometry, Fluorescence / methods
  • Surface Plasmon Resonance / methods
  • Tandem Repeat Sequences
  • Time Factors

Substances

  • Connectin
  • Immunoglobulins
  • Muscle Proteins
  • Recombinant Fusion Proteins
  • TTN protein, human
  • Gold
  • O(6)-Methylguanine-DNA Methyltransferase
  • Glutathione Transferase
  • Protein Kinases