Overexpression of acetohydroxyacid synthase from arabidopsis as an inducible fusion protein in Escherichia coli: Production of polyclonal antibodies, and immunological characterization of the enzyme

Bijay Singh, Gail Schmitt, Marcella Lillis, J. Mark Hand, Rajeev Misra

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) is the first enzyme unique to the biosynthesis of valine, leucine, and isoleucine. This enzyme is the target site of several classes of structurally unrelated herbicides. The conventional method of antibody production using purified protein has not been successful with this enzyme. Two separate fragments of a gene encoding a portion of the mature region of AHAS from Arabidopsis were fused with the trpE gene from Escherichia coli using the pATH1 vector. E. coli cells transformed with each respective plasmid expressed a fusion protein at levels greater than 10% of the total cell protein. The fusion protein was purified and used to immunize rabbits. Antisera obtained from the immunized rabbits immunoprecipitated AHAS activity from Arabidopsis cell free extracts. The anti-AHAS antisera reacted with a 65 kilodalton protein band in electrophoretically resolved extracts of Arabidopsis. In cross-reactivity tests, this antibody was able to immunoprecipitate AHAS activity from various plant species. Furthermore, a protein band with a molecular mass of 65 kilodaltons was detected in the crude extracts of all plant species tested on a Western blot. These results indicate that the 65 kilodalton protein represents AHAS in various plant species. The wide spectrum of cross-reactivity for the antisera supports the view that the AHAS enzyme is highly conserved across all plant species.

Original languageEnglish (US)
Pages (from-to)657-662
Number of pages6
JournalPlant Physiology
Volume97
Issue number2
DOIs
StatePublished - Jan 1 1991
Externally publishedYes

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science

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