[Extraction and purification of non-receptor type PTKs-Syk].
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.
Shan
Bao-en
BE
Research Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China. baoenshan@yahoo.com.cn
Dong
Qing
Q
Li
Hong-fen
HF
Chen
Jing
J
Dong
Jin-zhuo
JZ
Ma
Hong
H
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
D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase