Document Detail


Protease domain of human ADAM33 produced by Drosophila S2 cells.
MedLine Citation:
PMID:  15555945     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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].
Authors:
Winifred W Prosise; Taisa Yarosh-Tomaine; Zia Lozewski; Richard N Ingram; Jun Zou; Jian-Jun Liu; Feng Zhu; S Shane Taremi; Hung V Le; Wenyan Wang
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Protein expression and purification     Volume:  38     ISSN:  1046-5928     ISO Abbreviation:  Protein Expr. Purif.     Publication Date:  2004 Dec 
Date Detail:
Created Date:  2004-11-23     Completed Date:  2005-04-08     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  9101496     Medline TA:  Protein Expr Purif     Country:  United States    
Other Details:
Languages:  eng     Pagination:  292-301     Citation Subset:  IM    
Affiliation:
Structural Chemistry Department, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
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MeSH Terms
Descriptor/Qualifier:
ADAM Proteins
Animals
Cadmium Chloride / chemistry
Catalysis
Cell Line
Cloning, Molecular
Copper Sulfate / chemistry
Drosophila
Gene Expression Regulation, Enzymologic
Genetic Vectors / genetics
Glycosylation
Humans
Metalloendopeptidases / biosynthesis*,  genetics*,  isolation & purification
Mutation
Promoter Regions, Genetic
Protein Structure, Tertiary
Recombinant Fusion Proteins / biosynthesis,  genetics,  isolation & purification
Zinc / chemistry
Chemical
Reg. No./Substance:
0/Recombinant Fusion Proteins; 10108-64-2/Cadmium Chloride; 7440-66-6/Zinc; 7758-98-7/Copper Sulfate; EC 3.4.24.-/ADAM Proteins; EC 3.4.24.-/ADAM33 protein, human; EC 3.4.24.-/Metalloendopeptidases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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