Analysis of yeast protein kinases using protein chips

Nat Genet. 2000 Nov;26(3):283-9. doi: 10.1038/81576.

Abstract

We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae. Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides. The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples. Only small amounts of protein are required. Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips. We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine. The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region. Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Equipment Design
  • Evolution, Molecular
  • Fungal Proteins / analysis*
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism
  • Gene Expression Profiling / instrumentation*
  • Gene Expression Profiling / methods
  • Gene Expression Regulation, Enzymologic
  • Gene Expression Regulation, Fungal
  • Peptides / metabolism
  • Phosphorus Radioisotopes / analysis
  • Phosphorylation
  • Protein Conformation
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Protein-Tyrosine Kinases / analysis*
  • Protein-Tyrosine Kinases / chemistry
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / isolation & purification
  • Protein-Tyrosine Kinases / metabolism
  • Proteins / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Sequence Alignment
  • Sequence Analysis, Protein
  • Substrate Specificity

Substances

  • Fungal Proteins
  • Peptides
  • Phosphorus Radioisotopes
  • Proteins
  • Recombinant Fusion Proteins
  • poly(tyrosyl-glutamic acid)
  • Adenosine Triphosphate
  • Protein-Tyrosine Kinases