Expression of biologically active recombinant antifreeze protein His-MpAFP149 from the desert beetle (Microdera punctipennis dzungarica) in Escherichia coli.
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.
Qiu
Liming
L
Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, 14 Shengli Road, 830046, Urumqi, China.
Wang
Yan
Y
Wang
Jing
J
Zhang
Fuchun
F
Ma
Ji
J
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D001517:Coleoptera / Q000378:metabolism*; D019610:Cytoprotection; D003889:Desert Climate; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010802:Phylogeny; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012996:Solutions; D040681:Structural Homology, Protein; D013696:Temperature
D021301:Antifreeze Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D012996:Solutions; D006639:Histidine