[Extraction and purification of non-receptor type PTKs-Syk]

To extract and purify Syk protein from Sf21 cells transfected by Syk gene.

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.

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.

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.