PMID- 10648173
OWN - NLM
STAT- MEDLINE
DCOM- 20000309
LR  - 20161124
IS  - 1046-5928 (Print)
IS  - 1046-5928 (Linking)
VI  - 18
IP  - 1
DP  - 2000 Feb
TI  - Purification and characterization of human Syk produced using a baculovirus
      expression system.
PG  - 86-94
AB  - The cytoplasmic tyrosine kinase p72syk (Syk) plays an essential role in signaling
      via a variety of immune and nonimmune cell receptors. Syk is activated in
      response to the engagement of the appropriate cell surface receptors and can
      phosphorylate downstream targets and recruit additional SH2-domain-containing
      proteins. In order to study the characteristics of Syk in vitro, we have
      overexpressed untagged, full-length human Syk in a recombinant baculovirus
      expression system. The enzyme was purified to 95% purity using a novel two-step
      affinity chromatography process using reactive yellow and phosphotyrosine
      columns. Yields of 3-10 mg purified Syk were obtained from 1 liter of infected
      insect cells. Western blotting, internal protein sequencing, and the specific
      tyrosine phosphorylation of a Syk peptide substrate indicated authenticity of the
      purified protein. The enzymatic properties of Syk were in good agreement with
      published data for the human enzyme, as the apparent K(m) of Syk for ATP was 10
      microM and the peptide substrate was 3 microM. The recombinant protein also
      showed similar biochemical characteristics to the native protein isolated from
      B-cells such as autophosphorylation. Proteolytic cleavage of purified recombinant
      Syk was used to generate the kinase domain by micro-calpain. We therefore
      describe an efficient expression system and purification methodology to produce
      biologically active human Syk.
CI  - Copyright 2000 Academic Press.
FAU - Baldock, D
AU  - Baldock D
AD  - Respiratory Disease Therapeutic Area, Novartis Horsham Research Centre,
      Wimblehurst Road, Horsham, West Sussex, RH12 5AB, United Kingdom.
      Darren.Baldock@pharma.novartis.com
FAU - Graham, B
AU  - Graham B
FAU - Akhlaq, M
AU  - Akhlaq M
FAU - Graff, P
AU  - Graff P
FAU - Jones, C E
AU  - Jones CE
FAU - Menear, K
AU  - Menear K
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (Enzyme Precursors)
RN  - 0 (Intracellular Signaling Peptides and Proteins)
RN  - 0 (Recombinant Proteins)
RN  - EC 2.7.10.1 (Protein-Tyrosine Kinases)
RN  - EC 2.7.10.2 (SYK protein, human)
RN  - EC 2.7.10.2 (Syk Kinase)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Baculoviridae/genetics
MH  - Cell Line
MH  - Chromatography, Affinity
MH  - Enzyme Precursors/biosynthesis/*genetics/*isolation & purification
MH  - Gene Expression
MH  - Humans
MH  - Intracellular Signaling Peptides and Proteins
MH  - Kinetics
MH  - Molecular Sequence Data
MH  - Molecular Weight
MH  - Protein-Tyrosine Kinases/biosynthesis/*genetics/*isolation & purification
MH  - Recombinant Proteins/biosynthesis/genetics/isolation & purification
MH  - Spodoptera
MH  - Syk Kinase
EDAT- 2000/01/29 09:00
MHDA- 2000/03/11 09:00
CRDT- 2000/01/29 09:00
PHST- 2000/01/29 09:00 [pubmed]
PHST- 2000/03/11 09:00 [medline]
PHST- 2000/01/29 09:00 [entrez]
AID - 10.1006/prep.1999.1171 [doi]
AID - S1046-5928(99)91171-3 [pii]
PST - ppublish
SO  - Protein Expr Purif. 2000 Feb;18(1):86-94. doi: 10.1006/prep.1999.1171.