Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR - Université Denis Diderot - Paris VII
Article Dans Une Revue Plant Physiology Année : 2018

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR

Résumé

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b 6 f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast.
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Dates et versions

cea-01945922 , version 1 (04-09-2024)

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Louis Dumas, Francesca Zito, Pascaline Auroy, Xenie Johnson, Gilles Peltier, et al.. Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR. Plant Physiology, 2018, 177 (2), pp.465-475. ⟨10.1104/pp.17.01618⟩. ⟨cea-01945922⟩
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