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Expression, Purification and Characterization of Diguanylate Cyclase From Rhodococcus Ruber

Expression, Purification and Characterization of Diguanylate Cyclase From Rhodococcus Ruber

Sufang Kuang, Yuan Yuan, Zhonghao Wu, Ren Peng

Protein Expr Purif. 2019 Nov;163:105441.

PMID: 31195084

Abstract:

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-1. Some ions such as Zn2+, Mn2+, Fe2+, Ni2+ and Co2+ had inhibitory effects on the activity of the protein, while other ions such as Mg2+, K+ and Na+ slightly activated the protein. The fusion protein also showed rather high stability in the presence of toluene, cyclohexane and n-hexane.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42410998	Glutathione−Agarose			Price

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