Simplified purification of soluble histidine-tagged green fluorescent protein from cocoon of transgenic silkworm in metal affinity hydroxyapatite chromatography

Histidine-tagged proteins have been purified with immobilized nickel affinity chromatography. This method, however, bears some serious disadvantages, including carcinogenicity and potential leakage of the nickel and the handling and disposal costs. We developed a novel purification process for water-soluble histidine-tagged green fluorescent protein from the cocoons of transgenic silkworms, unlike conventional E. coli expression system which sometimes produced insoluble and inactive inclusion bodies. 60.3% extraction efficiency was achieved with 50 mM Tris containing 150 mM NaCl, pH 7.5. The extract was purified in one step on zinc-immobilized hydroxyapatite eluted with phosphate buffer. Purity was 91.2% with a recovery of 30.0 mu g from 160 mg of silk fiber. This purification method may provide a simple non-toxic alternative to nickel-immobilized metal affinity chromatography for purification of other histidine-tagged proteins. (C) 2010 Elsevier B.V. All rights reserved.