Detection and purification of backbone-cyclized proteins using a bacterially expressed anti-myc-tag single chain antibody

Sushma Chauhan, Chen Yuan Hou, Sang Taek Jung, Taek Jin Kang

Research output: Contribution to journalArticle

Abstract

A myc-tag and of which recognition by an antibody 9E10 has long been used for the detection and purification of recombinant proteins. We have previously expanded the application of the tag to the specific detection and purification of backbone-cyclized proteins. Here we sought a more practical way to using the 9E10 antibody by expressing its single chain antibody (scAb) form in Escherichia coli. The combined use of a strong T7 promoter and auto-induction strategy rather than early to mid-log induction of a Lac promoter resulted in the soluble over-expression of 9E10 scAb. However, the co-expression of a chaperone, Skp, was absolutely necessary for the activity even when the protein was expressed in a soluble manner. We could purify about 4 mg of 9E10 scAb from 1 l of culture, and the resulting scAb could be used to detect and purify the backbone-cyclized protein as the parental full-length 9E10. Moreover, the immunoaffinity resin prepared using 9E10 scAb could be regenerated several times after the elution of bound proteins using an acid, which added more value to the ready preparation of the active antibody in bacteria.

Original languageEnglish
Pages (from-to)38-44
Number of pages7
JournalAnalytical Biochemistry
Volume532
DOIs
Publication statusPublished - 2017 Sep 1
Externally publishedYes

Keywords

  • Immunodetection
  • Immunopurification
  • Myc-tag
  • Protein cyclization
  • Single chain antibody

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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