Screening and Analyzing of gp150-interacting Proteins of Dictyostelium discoideum

Abstract: In order to screen the interacting proteins of gp150， protein gp150 (lagC) C-terminal was expressed and polyclonal antibody of gp150 C-terminal was prepared and identified. The recombinant plasmid pET- 32a(+) with lagC C-terminal and 6-His tag was expressed in E. coli BL21 (DE3) host cells. The fusion protein with 6×His was purified by Ni2 + affinity chromatography column and then was used to immune the New Zealand rabbits for preparing polyclonal antibody. The titer and specificity of the rabbit’s antiserum were measured by ELISA and Western blot， respectively. The results showed that the fusion protein was successfully expressed and polyclonal antibody was also successfully obtained. The specificity of antibody was proved by Western blot analysis of expression product of gp150. The potency of the polyclonal antibody was as high as 1:128 000. The polyclonal antibody was used in co-immunoprecipitation and two gp150-intertacting proteins were captured and identified as 40S ribosomal protein S3 and 40S ribosomal protein S24 by mass spectrometer， which play important roles in regulating of cell cycle， cell development and apoptosis. The data suggest that gp150 can interact with RPS3 and RPS24 for regulating cell differentiation and development during multicellular development of Dictyostelium discoideum.

CSTR: 32200.14.cjcb.2010.03.0019