Efficient production of the C-terminal domain of secretory leukoprotease inhibitor as a thrombin-cleavable fusion protein in Escherichia coli |
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Authors: | Masuda, Ken-ichi Kamimura, Takashi Kanesaki, Masaharu Ishii, Koji Imaizumi, Atsushi Sugiyama, Takashi Suzuki, Yoji Ohtsuka, Eiko |
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Affiliation: | 1Teljin Institute for Biomedical Research 4-3-2 Asahi-ga-oka, Hino, Tokyo 191 3Faculty of Pharmaceutical Sciences, Hokkaido University Sapporo 060, Japan |
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Abstract: | We have developed a high-level production system for the C-terminaldomain of secretory leukoprotease inhibitor (SLPI) to investigateits pharmacological activities. A gene for the C-terminal domainof SLPI, (Asn55-AlalO7)SLPI, was constructed from chemicallysynthesized deoxyoligonucleotides. It was fused to a gene forthe N-terminal portion of human growth hormone via a DNA sequenceencoding Leu-Val-Pro-Arg, which can he cleaved by thrombin.The fused gene was expressed in Escherichia coli under the controlof a trp promoter, and the fusion protein was obtained as aninclusion body. After sulfonation of the cysteine residues,the sulfonated fusion protein was cleaved at the desired siteby thrombin. Sulfonated (Asn55-Ala107)SLPI was refolded in Trisbuffer containing reduced and oxidized glutathione. The resulting(Asn55-Ala107) SLPI was purified by cation-exchange chromatographyand reverse-phase high performance liquid chromatography. Thefinal yield was 50 mg/l culture. (Asn55-Ala107)SLPI was as activeagainst elastase as, but had less trypsin inhibitory activitythan, native SLPI. This system is suitable for the large-scaleproduction of the C-terminal domain of SLPI, which is an elastase-specificinhibitor. |
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Keywords: | domain structure/ E.coli/ expression/ fusion protein/ protease inhibitor |
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