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Plant Molecular Biology Reporter

, Volume 10, Issue 2, pp 199–206 | Cite as

Cloning of plant cDNAs encoding calmodulin-binding proteins using35S-labeled recombinant calmodulin as a probe

  • Hillel Fromm
  • Nam-Hai Chua
Protocol

Abstract

Radiolabelled calmodulin has previously been used to screen cDNA expression libraries to isolate calmodulin-binding proteins. We have modified this technique for the isolation of plant calmodulin-binding proteins. [35S]-methionine was used instead of the inorganic [35S]-sulfate, or125I used in previous methods. In addition, theE. coli pET expression system was chosen to obtain high levels of recombinant calmodulin at the time of labelling. The procedure thus takes into account both the specific activity of the probe and the amount of protein necessary for screening a large number of filters. Here we describe in detail a procedure for the production and purification of [35S]-recombinant calmodulin and the use of the radiolabelled protein as a probe to screen plant cDNA expression libraries. The [35S]-labeled calmodulin probe easily detects the λICM-1 phage encoding a partial mouse calmodulin-dependent protein kinase II that was previously isolated using a [125I]-calmodulin probe (Sikela and Hahn, 1987). Subsequently, a tobacco root cDNA expression library was screened and a positive clone encoding a calcium-dependent calmodulin-binding protein was isolated.

Key words

calcium signal transduction expression library screening 

Abbreviations

CAM

calmodulin

IPTG

isopropyl-β-D-thiogalactopyranoside

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Copyright information

© Kluwer Academic Publishers 1992

Authors and Affiliations

  • Hillel Fromm
    • 1
  • Nam-Hai Chua
    • 1
  1. 1.Laboratory of Plant Molecular BiologyThe Rockefeller UniversityNew YorkUSA

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