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Ancestral mutations as a tool for solubilizing proteins: The case of a hydrophobic phosphate-binding protein.

ABSTRACT: Stable and soluble proteins are ideal candidates for functional and structural studies. Unfortunately, some proteins or enzymes can be difficult to isolate, being sometimes poorly expressed in heterologous systems, insoluble and/or unstable. Numerous methods have been developed to address these issues, from the screening of various expression systems to the modification of the target protein itself. Here we use a hydrophobic, aggregation-prone, phosphate-binding protein (HPBP) as a case study. We describe a simple and fast method that selectively uses ancestral mutations to generate a soluble, stable and functional variant of the target protein, here named sHPBP. This variant is highly expressed in Escherichia coli, is easily purified and its structure was solved at much higher resolution than its wild-type progenitor (1.3 versus 1.9 Å, respectively).

SUBMITTER: Gonzalez D

PROVIDER: S-EPMC3907688 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Ancestral mutations as a tool for solubilizing proteins: The case of a hydrophobic phosphate-binding protein.

Gonzalez Daniel D Hiblot Julien J Darbinian Nune N Miller Jernelle C JC Gotthard Guillaume G Amini Shohreh S Chabriere Eric E Elias Mikael M

FEBS open bio 20140103

Stable and soluble proteins are ideal candidates for functional and structural studies. Unfortunately, some proteins or enzymes can be difficult to isolate, being sometimes poorly expressed in heterologous systems, insoluble and/or unstable. Numerous methods have been developed to address these issues, from the screening of various expression systems to the modification of the target protein itself. Here we use a hydrophobic, aggregation-prone, phosphate-binding protein (HPBP) as a case study. W ...[more]

PMID: 24490136

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Project description:Phosphorus (P) deficiency is a common problem in croplands where phosphate-based fertilizers are regularly used to maintain bioavailable P for plants. However, due to their limited mobility in the soil, there has been an increased interest in microorganisms that can convert insoluble P into a bioavailable form, and their use to develop phosphate-solubilizing bioinoculants as an alternative to the conventional use of P fertilizers. In this study, we proposed two independent experiments and explored two entirely different habitats to trap phosphate-solubilizing bacteria (PSBs). In the first experiment, PSBs were isolated from the rhizoplane of native plant species grown in a rock-phosphate (RP) mining area. A subset of 24 bacterial isolates from 210 rhizoplane morphotypes was selected for the inorganic phosphate solubilizing activities using tricalcium phosphate (TCP) as the sole P source. In the second experiment, we proposed an innovative experimental setup to select mycohyphospheric bacteria associated to arbuscular mycorrhizal fungal hyphae, indigenous of soils where agronomic plant have been grown and trapped in membrane bag filled with RP. A subset of 25 bacterial isolates from 44 mycohyphospheric morphotypes was tested for P solubilizing activities. These two bacterial subsets were then screened for additional plant growth-promoting (PGP) traits, and 16S rDNA sequencing was performed for their identification. Overall, the two isolation experiments resulted in diverse phylogenetic affiliations of the PSB collection, showing only 4 genera (24%) and 5 species (17%) shared between the two communities, thus underlining the value of the two protocols, including the innovative mycohyphospheric isolate selection method, for selecting a greater biodiversity of cultivable PSB. All the rhizoplane and mycohyphospheric PSB were positive for ammonia production. Indol-3-acetic acid (IAA) production was observed for 13 and 20 isolates, respectively among rhizoplane and mycohyphospheric PSB, ranging, respectively, from 32.52 to 330.27 μg mL-1 and from 41.4 to 963.9 μg mL-1. Only five rhizoplane and 12 mycohyphospheric isolates were positively screened for N2 fixation. Four rhizoplane PSB were identified as siderophore producers, while none of the mycohyphospheric isolates were. The phenotype of one PSB rhizoplane isolate, assigned to Pseudomonas, showed four additive PGP activities. Some bacterial strains belonging to the dominant genera Bacillus and Pseudomonas could be considered potential candidates for further formulation of biofertilizer in order to develop bioinoculant consortia that promote plant P nutrition and growth in RP-enriched soils.

Dataset Information

Ancestral mutations as a tool for solubilizing proteins: The case of a hydrophobic phosphate-binding protein.

Publications

Ancestral mutations as a tool for solubilizing proteins: The case of a hydrophobic phosphate-binding protein.

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