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  • Title: Expression of biologically active recombinant antifreeze protein His-MpAFP149 from the desert beetle (Microdera punctipennis dzungarica) in Escherichia coli.
    Author: Qiu L, Wang Y, Wang J, Zhang F, Ma J.
    Journal: Mol Biol Rep; 2010 Apr; 37(4):1725-32. PubMed ID: 19562508.
    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 degrees C at 0.09 mg/ml, and the supercooling point was -9.6 degrees C at 0.03 mg/ml. In vitro antifreeze activity assay by measuring the survival rate of bacteria at -7 and -20 degrees 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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