PMID- 9631522
OWN - NLM
STAT- MEDLINE
DCOM- 19980827
LR  - 20161124
IS  - 1046-5928 (Print)
IS  - 1046-5928 (Linking)
VI  - 13
IP  - 1
DP  - 1998 Jun
TI  - Expression and purification of correctly processed, active human TACE catalytic 
      domain in Saccharomyces cerevisiae.
PG  - 104-10
AB  - 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.
FAU - Clarke, H R
AU  - Clarke HR
AD  - Immunex Corporation, Seattle, Washington 98101, USA. clarkeh@immunex.com
FAU - Wolfson, M F
AU  - Wolfson MF
FAU - Rauch, C T
AU  - Rauch CT
FAU - Castner, B J
AU  - Castner BJ
FAU - Huang, C P
AU  - Huang CP
FAU - Gerhart, M J
AU  - Gerhart MJ
FAU - Johnson, R S
AU  - Johnson RS
FAU - Cerretti, D P
AU  - Cerretti DP
FAU - Paxton, R J
AU  - Paxton RJ
FAU - Price, V L
AU  - Price VL
FAU - Black, R A
AU  - Black RA
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (DNA Primers)
RN  - 0 (Oligopeptides)
RN  - 0 (Peptides)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - 98849-88-8 (FLAG peptide)
RN  - EC 3.4.24.- (ADAM Proteins)
RN  - EC 3.4.24.- (Metalloendopeptidases)
RN  - EC 3.4.24.86 (ADAM17 Protein)
RN  - EC 3.4.24.86 (ADAM17 protein, human)
SB  - IM
MH  - ADAM Proteins
MH  - ADAM17 Protein
MH  - Amino Acid Sequence
MH  - Base Sequence
MH  - Catalysis
MH  - DNA Primers
MH  - Glycosylation
MH  - Humans
MH  - Mass Spectrometry
MH  - Metalloendopeptidases/*genetics/isolation & purification/metabolism
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Oligopeptides
MH  - Peptides/isolation & purification
MH  - Recombinant Proteins/genetics/isolation & purification/metabolism
MH  - Saccharomyces cerevisiae/*genetics
MH  - Tumor Necrosis Factor-alpha/*metabolism
EDAT- 1998/06/19 00:00
MHDA- 1998/06/19 00:01
CRDT- 1998/06/19 00:00
PHST- 1998/06/19 00:00 [pubmed]
PHST- 1998/06/19 00:01 [medline]
PHST- 1998/06/19 00:00 [entrez]
AID - S1046-5928(98)90861-0 [pii]
AID - 10.1006/prep.1998.0861 [doi]
PST - ppublish
SO  - Protein Expr Purif. 1998 Jun;13(1):104-10. doi: 10.1006/prep.1998.0861.