title: Expression and purification of correctly processed, active human TACE catalytic domain in Saccharomyces cerevisiae.
abstract: Human tumor necrosis factor-alpha (TNF alpha) converting enzyme (TACE) releases soluble TNF alpha from cells. It is a member of the adamalysin family of metalloproteases. A truncated form of TACE cDNA was expressed in Saccharomyces cerevisiae and purified to homogeneity in order to study TACE structure and function. Recombinant TACE was expressed as a preproprotein including the pro- and catalytic (PROCAT) domains fused to the yeast alpha-factor leader. A C-terminal immunoreactive FLAG peptide was added for Western blot detection and anti-FLAG antibody column purification. We constructed two glycosylation mutant PROCAT TACE isoforms to facilitate purification. A PROCAT isoform, mutated to eliminate two N-linked glycosylation sites, was buffer exchanged and purified to homogeneity by ion exchange chromatography and an anti-FLAG antibody affinity step. N-terminal sequence analysis showed that the mutant preproprotein was processed in yeast at the furin protease cleavage site and yielded an active catalytic domain which has TNF alpha peptide-specific protease activity. Mass spectrometry of the purified catalytic domain showed that removal of both N-linked sites results in a homogeneous sized polypeptide lacking further posttranslational modifications.
authors.affiliation: Immunex Corporation, Seattle, Washington 98101, USA. clarkeh@immunex.com
authors.forename: H R
authors.initials: HR
authors.lastname: Clarke
authors.forename: M F
authors.initials: MF
authors.lastname: Wolfson
authors.forename: C T
authors.initials: CT
authors.lastname: Rauch
authors.forename: B J
authors.initials: BJ
authors.lastname: Castner
authors.forename: C P
authors.initials: CP
authors.lastname: Huang
authors.forename: M J
authors.initials: MJ
authors.lastname: Gerhart
authors.forename: R S
authors.initials: RS
authors.lastname: Johnson
authors.forename: D P
authors.initials: DP
authors.lastname: Cerretti
authors.forename: R J
authors.initials: RJ
authors.lastname: Paxton
authors.forename: V L
authors.initials: VL
authors.lastname: Price
authors.forename: R A
authors.initials: RA
authors.lastname: Black
mesh_terms: D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010455:Peptides / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C085706:FLAG peptide; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human