Gene cloning and prokaryotic expression of GDP-mannose 4,6-dehydratase from Nostoc fl agelliforme

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  • (School of Food and Biological Engineering, Shaanxi University of Science and Technology Xi’an 710021)

Online published: 2018-02-02

Abstract

Nostoc flagelliforme is a terrestrial cyanobacteria with stress-tolerance, which lives in poor condition. And the extracellular polysaccharide produced by Nostoc flagelliforme is proved to be with high edible and medicinal value. It was found in the previous work that the transcription level of gene encoded for GDP-mannose 4,6-dehydratase increased 2.18 times in Nostoc flagelliforme cultured under high salt concentration contrast to the control by RNA-Seq technology. Based on the transcriptome sequencing result and the homologous blast, the primer was designed  and the genome of Nostoc flagelliforme was utilized as template to clone the gene encoded for GDP-4,6-mannose dehydratase. The sequence with the size of 1080 bp was successfully obtained. According to the bioinformatical analysis, this gene was highly conserved, and the translated protein is hydrophilic and mainly consists of random coiling and alpha helix. The number of phosphorylation sites of tyrosine, threonine, and serine was 13, 15, and 17, respectively. The molecular weight of the protein is 41.08 kDa and the isoelectric point is 5.73.The amino acid residues with positive and negative charges are 38 and 46 respectively. Then, the recombinant expression plasmid was constructed by inserting the gene into the plasmid, Pet28a and successfully introduced into the competent Escherichia coli BL21 for prokaryotic expression. The expected size of recombinant protein (41.08 kDa) was obtained after 20 hours of induction with 1 mmol/L IPTG in 0.8 OD value at 16 degree. In this work, the gene for GDP-4,6-mannose dehydratase from Nostoc flagelliforme was successfully cloned and expressed in Escherichia coli for the first time. The result may lay a foundation for further study on molecular mechanism of polysaccharide metabolism from N. flagelliforme and provide theoretical basis for the construction of engineered strain for GDP-4,6-mannose dehydratase in the future.

Cite this article

CAI Guo-qiang et al . Gene cloning and prokaryotic expression of GDP-mannose 4,6-dehydratase from Nostoc fl agelliforme[J]. Food and Fermentation Industries, 2018 , 44(1) : 31 . DOI: 10.13995/j.cnki.11-1802/ts.015667

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