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Expression of biologically active recombinant antifreeze protein His-MpAFP149 from the desert beetle (Microdera punctipennis dzungarica) in Escherichia coli

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Abstract

An insect antifreeze protein gene Mpafp149 was cloned by the RT-PCR approach from the desert beetle Microdera punctipennis dzungarica. Sequence analysis revealed that this gene encoding a protein of 120 amid acids and this protein showed 65–76% homology with other insect antifreeze proteins, the deduced amino acid sequence displays very high similarities in those regions that contain tandem the 12-residue repeats (TCTxSxxCxxAx) domain and the TCT motif. Mpafp149 gene was cloned into pET-28a vector and expressed in Escherichia coli. A single-step purification based on specific binding of histidine residues was achieved. The purified His-MpAFP149 was SDS–PAGE analyzed, showing an atypical migration with molecular weight of about 24 kDa. The expression of His-MpAFP149 was confirmed by Western blot with specific binding to anti-GST-MpAFP149 antibody. The thermal hysteresis activity of the purified recombinant protein was 0.915°C at 0.09 mg/ml, and the supercooling point was −9.6°C at 0.03 mg/ml. In vitro antifreeze activity assay by measuring the survival rate of bacteria at −7 and −20°C respectively, with the protection of His-MpAFP149 showed that the His-MpAFP149 fusion protein was able to enhance the freeze resistance of bacteria.

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Acknowledgments

The authors are grateful to Gan Zhao for the gene cloning. This work was supported by a grant from the National Natural Science Foundation of China (No.30760056).

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Authors and Affiliations

  1. Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, 14 Shengli Road, 830046, Urumqi, China

    Liming Qiu, Yan Wang, Jing Wang, Fuchun Zhang & Ji Ma

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  1. Liming Qiu
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  2. Yan Wang
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  3. Jing Wang
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  4. Fuchun Zhang
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  5. Ji Ma
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Correspondence to Ji Ma.

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Qiu, L., Wang, Y., Wang, J. et al. Expression of biologically active recombinant antifreeze protein His-MpAFP149 from the desert beetle (Microdera punctipennis dzungarica) in Escherichia coli . Mol Biol Rep 37, 1725–1732 (2010). https://doi.org/10.1007/s11033-009-9594-3

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  • DOI: https://doi.org/10.1007/s11033-009-9594-3

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