Comparison of expression, purification and characterization of a new pectate lyase from Phytophthora capsici using two different methods.

Huizheng Wang, Li Fu, Xiuguo Zhang

Department of Plant Pathology, Shandong Agricultural University, Tai'an, 271018, Shandong, China.

BMC biotechnology 2011

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Pectate lyases (PELs) play an important role in the infection process of plant pathogens and also have a commercial significance in industrial applications. Most of the PELs were expressed as soluble recombinant proteins, while a few recombinant proteins were insoluble. The production of a large-scale soluble recombinant PEL would allow not only a more detailed structural and functional characterization of this enzyme but also may have important applications in the food industry. We cloned a new pectate lyase gene (Pcpel2) from Phytophthora capsici. Pcpel2 was constructed by pET system and pMAL system, and both constructs were used to express the PCPEL2 in Escherichia coli BL21 (DE3) pLysS. The expressed products were purified using affinity chromatography and gel filtration chromatography. The purity, specific activity and pathogenicity of the purified PCPEL2 expressed by the pMAL system were higher than the purified PCPEL2 expressed by the pET system. In addition, some other characteristics of the purified PCPEL2 differed from the two systems, such as crystallographic features. Purified PCPEL2 expressed by the pMAL system was crystallized by the hanging-drop vapour-diffusion method at 289 K, and initial crystals were grown. The two different methods and comparison presented here would be highly valuable in obtaining an ideal enzyme for the downstream experiments, and supply an useful alternative to purify some insoluble recombinant proteins.

Citation

Huizheng Wang, Li Fu, Xiuguo Zhang. Comparison of expression, purification and characterization of a new pectate lyase from Phytophthora capsici using two different methods. BMC biotechnology. 2011;11:32