Expression, purification and characterization of diguanylate cyclase from Rhodococcus ruber.

08:00 EDT 10th June 2019 | BioPortfolio

Summary of "Expression, purification and characterization of diguanylate cyclase from Rhodococcus ruber."

Diguanylate cyclases (DGCs) were responsible for the synthesis of second messenger cyclic di-guanosine monophosphate (c-di-GMP), which were involved in various physiological activities of bacterial species. Here, a full-length DGC from Rhodococcus ruber SD3 fused with glutathione-S-transferase (GST) was expressed in E. coli and purified by glutathione agarose resin. The apparent molecular mass of one subunit of the purified diguanylate cyclase with GST tag (GST-DGC) was estimated to be 71.9 kDa by SDS-PAGE, which was approximately in accordance with the theoretical value of 73.0 kDa. The sequence of GST-DGC was confirmed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The blue native PAGE indicated that GST-DGC formed octamer. The optimum pH and temperature for GST-DGC activity were 8.0 and 47 °C, respectively. The fusion protein exhibited high thermostability, and 94% of activity was retained when the protein was incubated at 87 °C for 1 h. Moreover, the fusion protein showed pH stability. The Km, Vmax and Kcat values for GST-DGC enzyme were 9.8 μM, 0.7 μM/min and 1.3 S. Some ions such as Zn, Mn, Fe, Ni and Co had inhibitory effects on the activity of the protein, while other ions such as Mg, K and Na slightly activated the protein. The fusion protein also showed rather high stability in the presence of toluene, cyclohexane and n-hexane.


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Name: Protein expression and purification
ISSN: 1096-0279


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