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      Positive Effects of Isopropanol as a Co-Precipitant in Glycerol-3-Phosphate Acyltransferase Crystallization

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          Abstract

          Glycerol-3-phosphate acyltransferase (GPAT) is considered as the rate-limiting enzyme of glycerolipid synthesis pathway and the core element in lysophosphatidic acid (LPA) synthesis. For understanding its catalytic mechanism, the structural biology study is expected, but is always hindered by obtaining crystals for X-ray diffraction analysis. In this study, a progressive strategy to optimize the crystal of microalgae plastidial GPAT was presented. After the expression and purification of GPAT, the crystals were screened by hanging-drop and only clusters were obtained. The crystals were optimized by adjusting temperature, pH, protein concentration, or precipitant, but little improvement. To improve the interaction between protein and precipitant, the isopropanol was applied as co-precipitant. The qualified crystals formed. It’s suggested that isopropanol is critical to affect protein crystallization by altering polyethylene glycol (PEG)-water-protein interaction when PEG serves as precipitant. The resulting crystal diffracted to a resolution of 2.75 Å and belonged to space group P1, with unit-cell parameters a = 50.79, b = 80.09, c = 88.21 Å, and α = 62.85, β = 73.04, γ = 80.53˚. This work introduced a new strategy to optimize the crystal of the heterogeneous catalysis enzymes like GPAT and provided the fundamental structural information for the oriented synthesis of marine microalgae glycerolipid.

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          Author and article information

          Journal
          JOUC
          Journal of Ocean University of China
          Science Press and Springer (China )
          1672-5182
          10 January 2019
          01 February 2019
          : 18
          : 1
          : 227-231
          Affiliations
          1 Marine Bioengineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
          2 University of Chinese Academy of Sciences, Beijing 100049, China
          Author notes
          *Corresponding author: XUE Song
          Article
          s11802-019-3519-0
          10.1007/s11802-019-3519-0
          Copyright © Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2019.

          The copyright to this article, including any graphic elements therein (e.g. illustrations, charts, moving images), is hereby assigned for good and valuable consideration to the editorial office of Journal of Ocean University of China, Science Press and Springer effective if and when the article is accepted for publication and to the extent assignable if assignability is restricted for by applicable law or regulations (e.g. for U.S. government or crown employees).

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          Self URI (journal-page): https://www.springer.com/journal/11802

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