Cloning of plant cDNAs encoding calmodulin-binding proteins using35S-labeled recombinant calmodulin as a probe |
| |
Authors: | Hillel Fromm Nam-Hai Chua |
| |
Institution: | (1) Laboratory of Plant Molecular Biology, The Rockefeller University, 10021 New York, NY, USA;(2) Present address: Department of Plant Genetics, The Weizmann Institute of Science, 76100 Rehovot, Israel |
| |
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. |
| |
Keywords: | calcium signal transduction expression library screening |
本文献已被 SpringerLink 等数据库收录! |
|