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Characterization and Expression of a GDSL-Like Lipase Gene from Brassica napus in Nicotiana benthamiana

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Abstract

The GDSL esterase and lipase families play important roles in abiotic stress, pathogen defense, seed development and lipid metabolism. Identifying the lipase activity of the putative GDSL lipase is the prerequisite for dissecting its function. According to the sequence similarity and the conserved domains, we cloned the Brassica napus BnGLIP gene, which encodes a GDSL-like protein. We failed to identify the BnGLIP lipase activity in the bacterium and yeast expression systems. In this paper, we expressed the BnGLIP gene by fusing a 6× His tag in Nicotiana benthamiana and purified the recombinant protein. The extraction buffer contained 1 % (v/v) n-caprylic acid and was able to remove most of the protein impurities. About 50 μg of recombinant BnGLIP was obtained from 1 g of N. benthamiana leaves. The lipase activity was tested with the purified BnGLIP and the maximum enzyme activity reached 17.7 mM/mg. In conclusion, this study found that the recombinant protein BnGLIP expressed in tobacco system was effectively purified and was detected as a GDSL lipase.

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Abbreviations

BnGLIP:

Braasican napus GDSL-like lipase

BnSCE3:

Brassica napus sinapine esterase

IgG:

Immunoglobulin G

IMAC:

Immobilized-metal affinity chromatography

PBS:

Phosphate buffer saline

ρ-NP:

ρ-Nitrophenol

ρ-NPL:

ρ-Nitrophenyl

CA:

Caprylic acid

Wb:

Western blot

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Acknowledgments

This work was supported by the Natural Science Foundation of China (31271760 and 31071672). We thank Professor George P. Lomonossoff at the John Innes Centre for providing the vector, pEAQ-HT-DEST1.

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Authors and Affiliations

  1. Institute of Life Sciences, Jiangsu University, Zhenjiang, 212013, People’s Republic of China

    Xiaoli Tan, Suzhen Yan, Renke Tan, Zhiyan Zhang, Zheng Wang & Jie Chen

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  1. Xiaoli Tan
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  2. Suzhen Yan
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  3. Renke Tan
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  4. Zhiyan Zhang
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  5. Zheng Wang
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  6. Jie Chen
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Correspondence to Xiaoli Tan or Suzhen Yan.

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Tan, X., Yan, S., Tan, R. et al. Characterization and Expression of a GDSL-Like Lipase Gene from Brassica napus in Nicotiana benthamiana . Protein J 33, 18–23 (2014). https://doi.org/10.1007/s10930-013-9532-z

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  • DOI: https://doi.org/10.1007/s10930-013-9532-z

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