Development and Applications of a Calmodulin-Based Fusion Protein System for the Expression and Purification of WW and Zinc Finger Modules

Calmodulin from Homo sapiens is an α-helical calcium-binding protein that expresses to high levels in E. coli. When the N-terminus of a calmodulin variant is bound to Ca2+, it undergoes a conformational change, exposing hydrophobic pockets. This property can be utilized for purification purposes, as these pockets bind to phenyl sepharose resin with high affinity. Washing with EDTA chelates the Ca2+ ions from the protein, inducing a conformational change back to the more folded state and eluting the protein from the column. We describe herein the use of a protein expression and purification technique using the calmodulin variant and a short linker for proteolytic cleavage by the mutant NIa-Pro tobacco etch virus protease. We have shown this approach to be useful in obtaining purified quantities of various small proteins that could not be expressed using other methods, including high enough concentrations of a designed WW domain protein for NMR structural analysis. We have also obtained promising results on the usefulness of this procedure to express and purify zinc finger proteins without the addition of zinc ions or other cofactors.

The Arabidopsis Cysteine-Rich GASA5 Is a Redox-Active Metalloprotein that Suppresses Gibberellin Responses

The plant-specific GIBBERELLIN （GA）-STIMULATED TRANSCRIPT 1 （GAST1）-like genes have been identified in numerous plant species and many of them are regulated by the phytohormone GA. All encode small proteins with a conserved cysteine-rich C-terminus, containing 12 cysteines in conserved positions. The fact that the number and positions of all 12 cysteines have not changed during the course of evolution strongly implies fundamental and important biochemical and bioloaical functions.

Gibberellin in tomato:metabolism,signaling and role in drought responses

The growth-promoting hormone gibberellin(GA)regulates numerous developmental processes throughout the plant life cycle.It also affects plant response to biotic and abiotic stresses.GA metabolism and signaling in tomato(Solanum lycopersicum)have been studied in the last three decades and major components of the pathways were characterized.These include major biosynthesis and catabolism enzymes and signaling components,such as the three GA receptors GIBBERELLIN INSENSITIVE DWARF 1(GID1)and DELLA protein PROCERA(PRO),the central response suppressor.The role of these components in tomato plant development and response to the environment have been investigated.Cultivated tomato,similar to many other crop plants,are susceptible to water deficiency.Numerous studies on tomato response to drought have been conducted,including the possible role of GA in tomato drought resistance.Most studies showed that reduced levels or activity of GA improves drought tolerance and drought avoidance.This review aims to provide an overview on GA biosynthesis and signaling in tomato,how drought affects these pathways and how changes in GA activity affect tomato plant response to water deficiency.It also presents the potential of using the GA pathway to generate drought-tolerant tomato plants with improved performance under both irrigation and water-limited conditions.