RRC ID 37011
Author Scavuzzo-Duggan TR, Chaves AM, Roberts AW.
Title A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae).
Journal Appl Plant Sci
Abstract PREMISE OF THE STUDY:A method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens.
METHODS AND RESULTS:A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins.
CONCLUSIONS:Compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.
Volume 3(7)
Published 2015-7
DOI 10.3732/apps.1500023
PII apps1500023
PMID 26191463
PMC PMC4504723
IF 1.187
Resource
Arabidopsis / Cultured plant cells, genes pdp24095