title: [Extraction and purification of non-receptor type PTKs-Syk].
abstract: OBJECTIVE
  To extract and purify Syk protein from Sf21 cells transfected by Syk gene.
  
  
  METHODS
  Sf21 cells were transfected with recombinant syk gene. After 48 h of incubation at 28 degrees Celsius, the transfected cells were collected and sonicated with Sonfier sonicator on ice. Filtered cell extract was loaded onto a Reactive Yellow-3 resin column and Toyopearl AF-Heparin-650 M column respectively. The characters of Syk protein in the fractions were identified by SDS-PAGE, Western blotting and IEF.
  
  
  RESULTS
  225 mg of protein containing Syk were obtained from Sf21 cells (2.5 x 10(9))extract. There were two subpopulations in the elution of Reactive Yellow-3 resin column with the same relative molecular mass (Mr) 72 x 10(3). The two subpopulations were then applied on Toyopearl AF-Heparin-650 M column and two pure proteins were obtained. The results of SDS-PAGE, Western blotting, and IEF showed the two proteins having the same relative molecular mass (72 x 10(3)), corresponding to Syk, but with different pI.
  
  
  CONCLUSIONS
  The yield of Syk was 8 mg from 2.5 billion cells and the purity was > 95%. The two purified Syk proteins have the same Mr and different pI. The purified Syk protein can be applied to study Syk's mechanism, produce anti-Syk antibody and invent Syk diagnosis kit, etc.
authors.affiliation: Research Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China. baoenshan@yahoo.com.cn
authors.forename: Bao-en
authors.initials: BE
authors.lastname: Shan
authors.forename: Qing
authors.initials: Q
authors.lastname: Dong
authors.forename: Hong-fen
authors.initials: HF
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Jin-zhuo
authors.initials: JZ
authors.lastname: Dong
authors.forename: Hong
authors.initials: H
authors.lastname: Ma
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000302:isolation & purification*; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology*; D000072377:Syk Kinase; D014162:Transfection
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase