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 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 ( approximately 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].
Prosise
Winifred W
WW
Structural Chemistry Department, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
Yarosh-Tomaine
Taisa
T
Lozewski
Zia
Z
Ingram
Richard N
RN
Zou
Jun
J
Liu
Jian-Jun
JJ
Zhu
Feng
F
Taremi
S Shane
SS
Le
Hung V
HV
Wang
Wenyan
W
D051722:ADAM Proteins; D000818:Animals; D019256:Cadmium Chloride / Q000737:chemistry; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D019327:Copper Sulfate / Q000737:chemistry; D004330:Drosophila; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D009154:Mutation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015032:Zinc / Q000737:chemistry
D011993:Recombinant Fusion Proteins; D051722:ADAM Proteins; C450441:ADAM33 protein, human; D008666:Metalloendopeptidases; D015032:Zinc; D019256:Cadmium Chloride; D019327:Copper Sulfate