Monocrystal Culture and Crystallization Conditions Optimization of HspB from Pseudomonas putida

doi:10.13560/j.cnki.biotech.bull.1985.2015.11.031

Abstract

Abstract:

It was to obtain the monocrystal of HspB, the key monooxygenase in the nicotine degradation pathway from Pseudomonas putida, for X-ray diffraction. The expression plasmid was constructed by site-directed mutagenesis PCR and was expressed in Escherichia coli. Target protein was purified with immobilized metal-chelating affinity chromatography, TEV protease digestion and gel filtration chromatography. Crystal culture though hanging-drop vapor-diffusion method. The recombinant plasmid was successfully constructed and expressed high. Compared to digestion on the column, digestion in the solution during dialysis is more efficient, and the purification strategy was determined to obtain HspB with high purity. The optimal crystallization condition was identified as 22% PEG3350, 0.1 mol/L Bis-Tris pH6.5, 0.21 mol/L MgCl₂, 18℃, seeding after orthogonal experiments. HspB with His-tag cleavaged can obtain a monocrystal which resolution reaches 1.8Å.

Key words: Pseudomonas putida, nicotine degradation, monooxygenase, site-directed mutagenesis, crystallization

Wu Zhijie, Wu Geng, Tang Hongzhi, Xu Ping. Monocrystal Culture and Crystallization Conditions Optimization of HspB from Pseudomonas putida[J]. Biotechnology Bulletin, 2015, 31(11): 236-242.

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