压力循环技术(PCT)为弗兰克氏菌蛋白的研究提供方便

Frankia are gram-positive, filamentous, nitrogen-fixing actinobacteria that are symbiotic with over 200 different species of plants. Frankia produce three cell types: vegetative hyphae, spores located in sporangia, and the unique lipid-enveloped cellular structures, termed vesicles. Vesicles are formed inside plant cell nodules, or in culture under nitrogen limiting conditions, and act as specialized structures for the nitrogen fixation process. The mature vesicle is surrounded by an envelope that extends down the stalk of the vesicle past the basal septum, which separates the vesicle from the hyphae. Techniques have been developed for the isolation and urification of intact vesicles from Frankia grown in culture [1,2,3]. Initial investigations on the properties of purified vesicles have focused on nitrogen metabolism [3,4]. Protein extraction from vesicles has historically been difficult and a significant bottleneck to proteomic studies because vesicles are resilient structures that are difficult to disrupt. Reliable and comprehensive proteomic maps of Frankia hyphae and vesicles can only be assured when proteins are isolated reproducibly and in a manner in which they are accurately represented in the downstream analysis. For example, 2DGE can be an accurate representation of a proteome only if the entire protein constituency of cells is recovered during the sample preparation process. While French press treatment effectively lyses hyphae, it fails to disrupt Frankia vesicles. Pressure Cycling Technology (PCT) used in combination with a specialized lysis reagent effectively disrupted purified vesicles enabling further investigation of the proteome of the vesicles.