Development of INSOL-tag for proteome-wide protein handling and its application in protein array analysis

Genes Cells. 2020 Jan;25(1):41-53. doi: 10.1111/gtc.12735. Epub 2019 Dec 3.

Abstract

Proteomic analysis requires protein tags that enable high-throughput handling; however, versatile tags that can be used in in vitro expression systems are currently lacking. In this study, we developed an insoluble protein tag, INSOL-tag, derived from human transcription factor MafG. The INSOL-tagged target protein is expressed in a eukaryotic in vitro expression system and recovered as a pellet following centrifugation at 19,000 × g for 20 min. Comparisons of the target protein recovery rates of GST-tag and INSOL-tag using 111 cytoplasmic proteins revealed a fourfold increase in the yield of INSOL-tagged proteins. Using 267 cancer antigens purified with INSOL-tag, we subsequently developed an INSOL-CTA array method, for profiling autoantibodies in sera of cancer patients. The detection limit of the array was approximately 11.1 pg IgG, and the correlation with ELISA was high (R2 = .993, .955). Moreover, when autoantibody profiling of digestive cancer patient sera was performed, antigen spreading was observed. These data suggest that INSOL-tag is a versatile tag that can insolubilize a wide range of target proteins. It is therefore expected to become a powerful tool in comprehensive protein preparation for protein arrays, antibody production, and mass spectrometry.

Keywords: cancer antigens; protein array; protein tag; transcription factor MafG.

MeSH terms

  • High-Throughput Screening Assays / methods
  • Humans
  • MafG Transcription Factor / genetics
  • MafG Transcription Factor / isolation & purification*
  • MafG Transcription Factor / metabolism*
  • Mass Spectrometry / methods
  • Protein Array Analysis / methods
  • Protein Engineering / methods
  • Proteome / genetics
  • Proteomics / methods*
  • Repressor Proteins / genetics
  • Repressor Proteins / isolation & purification*
  • Repressor Proteins / metabolism*
  • Transcription Factors / metabolism

Substances

  • MAFG protein, human
  • MafG Transcription Factor
  • Proteome
  • Repressor Proteins
  • Transcription Factors