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GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

ABSTRACT: Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3) (-) as the first ionic interaction partner, but not necessarily for Ca(2+). The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

SUBMITTER: Weber E

PROVIDER: S-EPMC3463529 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

Weber Eva E Guth Christina C Weiss Ingrid M IM

PloS one 20121003 10

Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in ...[more]

PMID: 23056388

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Project description:Simple plugging of the high-permeability "thief zones" of oil reservoirs is the most plausible and also the most straightforwardly achievable approach to enhance sweep efficiency and oil recovery. Sporosarcina pasteurii is a representative microorganism with the ability to precipitate calcium carbonate (CaCO3) via enzymatic hydrolysis of urea in the presence of calcium ions. Microbially induced calcium carbonate precipitation (MICP) can cement and seal the granular and fractured media and thus can be used as a potential microbial plugging agent for the high-permeability zones of oil reservoirs. The following investigated the microscopic characteristics of MICP plugging and its efficacy in permeability reduction. The columns of near-spherical silica sand and angular silica sand with three separate granularities (40/60, 60/80, and 80/120 mesh) were used as artificial rock cores representing distinct pore sizes and pore characteristics to investigate the efficacy and microprocess of MICP plugging with different biotreatment periods. The results indicated that permeability is reduced significantly after only short periods of biotreatment. After eight cycles of MICP treatments, the permeability for each type of cores dropped by 54-90% of individual initial permeabilities. The measured CaCO3 content indicated that the decreasing rate in permeability with the increasing CaCO3 content experiences three contrasting stages, namely, slow decline, speedy decline, and plateauing. X-ray diffraction indicated that most of the generated CaCO3 crystals occur as vaterite with only a small amount of calcite. Imaging by scanning electron microscopy further revealed the microprocess of MICP plugging. Microorganisms first concentrate on the pore wall to secrete CaCO3, forming a thin and large uniform layer of CaCO3. Then, some nucleation sites of CaCO3 crystals will experience further preferential growth, resulting in large, dominant crystals that act as a plugging agent within the pore space. Compared to extracellular polymeric substances, which are currently the primary microbial plugging agent used to enhance sweep efficiency of oil reservoirs, bio-CaCO3 appears more effective in plugging in terms of its morphology, size, and growth characteristics.

Dataset Information

GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

Publications

GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

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