Heterologous Expression of the Avirulence Gene Product, NIP1, from the Barley Pathogen Rhynchosporium secalis

Author： Gierlich A. van 't Slot K.A.E. Li V.M. Marie C. Hermann H. Knogge W.

ISSN： 1046-5928

Source： Protein Expression and Purification, Vol.17, Iss.1, 1999-10, pp. : 64-73

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Abstract

NIP1, the product of the avirulence gene AvrRrs1 from Rhynchosporium secalis, a fungal pathogen of barley, is a small secreted cysteine-rich protein. This protein is essential for the specific recognition of the fungus by host plants carrying the complementary resistance gene Rrs1. Different heterologous expression systems were tested to produce sufficient quantities of NIP1 to allow its utilization in receptor identification and isolation. In addition, protein amounts higher than those produced in fungal cultures are required to determine its 3D structure and to analyze its interaction with a receptor. The most efficient method, the synthesis of a His-tag fusion protein in Escherichia coli combined with a refolding procedure, yielded up to 3 mg of recombinant NIP1 from a 1-liter bacterial culture. After removal of the His-tag, the recombinant protein showed the same physicochemical characteristics as the native NIP1 and, most importantly, full biological activity.