Engineering of a Phosphorylatable Tag for Specific Protein Binding on Zirconium Phosphonate Based Microarrays

J Biol Inorg Chem. 2012 Mar;17(3):399-407. doi: 10.1007/s00775-011-0863-y. Epub 2011 Dec 13.

Abstract

A phosphorylatable tag was designed and fused at the C-terminal end of proteins, which allowed efficient and oriented immobilization of capture proteins on glass substrates coated with a zirconium phosphonate monolayer. The concept is demonstrated using Nanofitin directed against lysozyme. This peptide tag (DSDSSSEDE) contains four serines in an acidic environment, which favored its in vitro phosphorylation by casein kinase II. The resulting phosphate cluster at the C-terminal end of the protein provided a specific, irreversible, and multipoint attachment to the zirconium surface. In a microarray format, the high surface coverage led to high fluorescence signal after incubation with Alexa Fluor 647 labeled lysozyme. The detection sensitivity of the microarray for the labeled target was below 50 pM, owing to the exceptionally low background staining, which resulted in high fluorescence signal to noise ratios. The performance of this new anchoring strategy using a zirconium phosphonate modified surface compares favorably with that of other types of microarray substrates, such as nitrocellulose-based or epoxide slides, which bind proteins in a nonoriented way.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Enzyme-Linked Immunosorbent Assay
  • Glass / chemistry
  • Molecular Sequence Data
  • Organophosphonates / chemistry*
  • Organophosphonates / metabolism
  • Peptides / chemistry*
  • Peptides / genetics
  • Peptides / metabolism
  • Phosphorylation
  • Protein Array Analysis*
  • Protein Binding
  • Protein Engineering*
  • Spectrometry, Mass, Electrospray Ionization
  • Surface Properties
  • Zirconium / chemistry*
  • Zirconium / metabolism

Substances

  • Organophosphonates
  • Peptides
  • Zirconium