Prokaryotic expression, purification and characterization of arginine kinase of Bombyx mori.
- Author:
Huawei HE
1
;
Yejing WANG
2
;
Minjian ZHAO
2
;
Shuguang WEI
1
;
Peng ZHAO
1
;
Wenchao JIANG
1
;
Li'na LIU
1
;
Ping ZHAO
1
Author Information
- Publication Type:Journal Article
- Keywords: Bombyx mori; activity; arginine kinase; expression and purification; structure
- From: Chinese Journal of Biotechnology 2017;33(7):1109-1123
- CountryChina
- Language:Chinese
- Abstract: Arginine kinase (AK) is a key enzyme in energy metabolism of invertebrates and plays an important regulatory role in the life activities such as growth and development, nutrition utilization, immune resistance and stress response. Arginine kinase of Bombyx mori (BmAK) is related to the energy balance and anti-NPV process, but there is little research on its molecular structure and enzymatic properties. We cloned the ORF sequence of BmAK gene, and analyzed chromosomal localization, genomic structure, mRNA structure, secondary and tertiary structure. Phylogenetic analysis indicated that AK was highly conserved in evolution. Soluble recombinant BmAK was obtained by prokaryotic expression, and purified by Ni-NTA affinity chromatography. The circular dichroism spectroscopy showed that BmAK contained α-helix structures, and its α-helix structures were relatively stable in the pH range between 5 and 10. Enzyme activity analysis showed that the optimum temperature of BmAK was 30 ℃ and the optimum pH of BmAK was 7.5. The optimal temperature of BmAK was 25 ℃. Between 15 ℃ and 30 ℃, the structure and activity of BmAK was relatively stable. The structure of BmAK was relatively stable at pH 7.0. Our findings reveal the structure and function of BmAK to develop novel green safe and environmentally friendly insecticides.
