Protease domain of human ADAM33 produced by Drosophila S2 cells

Human ADAM33 is a multiple-domain, type-I transmembrane zinc metalloprotease recently implicated in asthma susceptibility [Nature 418 (2002) 426]. To provide an active protease for functional studies, expression of a recombinant ADAM33 zymogen (pro-catalytic domains, pro-CAT) was attempted in severa...

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Bibliographic Details
Published in:	Protein expression and purification Vol. 38; no. 2; pp. 292 - 301
Main Authors:	Prosise, Winifred W., Yarosh-Tomaine, Taisa, Lozewski, Zia, Ingram, Richard N., Zou, Jun, Liu, Jian-Jun, Zhu, Feng, Taremi, S. Shane, Le, Hung V., Wang, Wenyan
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-12-2004
Subjects:	ADAM Proteins ADAM33 Animals Cadmium Cadmium Chloride - chemistry Catalysis Catalytic domain Cell Line Cloning, Molecular Copper Copper Sulfate - chemistry Drosophila Drosophila S2 cells Gene Expression Regulation, Enzymologic Genetic Vectors - genetics Glycosylation Humans Metalloendopeptidases - biosynthesis Metalloendopeptidases - genetics Metalloendopeptidases - isolation & purification Metalloprotease Mutation PMT Promoter Regions, Genetic Protein Structure, Tertiary Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Zinc Zinc - chemistry Zymogen Zymogen Cadmium PMT ADAM33 Metalloprotease Catalytic domain Drosophila S2 cells Copper Zinc
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Summary:	Human ADAM33 is a multiple-domain, type-I transmembrane zinc metalloprotease recently implicated in asthma susceptibility [Nature 418 (2002) 426]. To provide an active protease for functional studies, expression of a recombinant ADAM33 zymogen (pro-catalytic domains, pro-CAT) was attempted in several insect cells. The pro-CAT was cloned into baculovirus under the regulation of the polyhedron promoter and using either the honeybee mellitin or ADAM33 signal sequence. Sf9 or Hi5 cells infected with these recombinant viruses expressed the majority of the protein unprocessed and as inclusion bodies (∼10 mg/L). On the other hand, similar constructs could be expressed, processed, and secreted by Drosophila S2 cells using a variety of constitutive (actin, pAc5.1) or inducible (metallothionein, PMT) promoters and leader sequences (e.g., native and BiP). Higher expression level of 10-fold was observed for the inducible system resulting in an average yield of 20 mg/L after purification. The majority of the catalytic domain purified from the Drosophila conditioned media remained associated with the pro-domain after several chromatography steps. An induction cocktail containing cadmium chloride and zinc chloride was subsequently developed for the PMT system as an alternative to using cupric sulfate or cadmium chloride as single inducers. The novel induction cocktail resulted in an increased ratio of secreted catalytic to pro-domain, and yielded milligram amounts of highly purified protease. The availability of this modified expression system facilitated purification of the wild type and several glycosylation mutants, one of which (N231Q) crystallized recently for X-ray structure determination [J. Mol. Biol. 335 (2003) 129].
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1046-5928 1096-0279
DOI:	10.1016/j.pep.2004.09.004