Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.

Identification of CTCL tumor antigens by SEREX / 解放军医学杂志

Objective To isolate and purify the membrane protein of SeAx cells, which was derived from cutaneous T-cell lymphoma cell line (CTCL), and to prepare polyclonal antibodies against membrane proteins. CTCL antigens were screened with SEREX method from the cDNA expression library of SeAx cells. Methods SeAx cells were cultured to 2?1010 and membrane proteins were purified by means of differential centrifugation after homogenate was made, and organelle protein specific antibodies were used to perform Western blot. Rabbit was immunized 4 times by the membrane proteins, and the serum antibody titer was measured by ELISA after each immunization. The specific interactions between antibodies in serum and SeAx proteins were detected by Western blotting. E. coli were initially transfected by recombinant phages, and the recombinant proteins expressed were transferred onto the nitrocellulose membranes, which were then again reacted with diluted serum from the immunized rabbit. Positive clones were subcloned and the nucleotide sequence of the cDNA inserts was determined. Finally, cDNA sequence analysis via database searching was performed to define the gene. Results Membrane proteins of SeAx cells were purified by differential centrifugation and a 1?10-5 titer of antibody was obtained after 4 times of immunization. 1?106 phage clones were screened, 25 of which were positive. Nucleotide sequencing and database searching showed that 4 cDNA inserts were cell membrane proteins, respectively as integrin alpha 4,ligatin, matrix metalloproteinase 24 and MHC-I molecule HLA-A. Conclusion Membrane proteins of CTCL and SeAx cells, are purified and polyclonal antibodies against SeAx cell membrane proteins are successfully prepared. The 4 genes of membrane proteins are identified by means of SEREX from SeAx cDNA library, expressed by phages. These genic candidates are able to induce systemic humoral immune reactions and might therefore be promising targets for immunotherapeutic interventions of CTCL.

Protein expression and preparation of polyclonal antibody of AD-004 and study on its expression in the adrenal and testis / 中华内分泌代谢杂志

Objective To prepare rabbit antibody against mouse AD-004 by AD-004 expressed in the prokaryotic expression system and to identify its distribution in the testis and adrenal. Methods The full-length cDNA of mouse AD-004 was cloned into PET28 plasmid, and the protein was induced in E. coli BL21 bacteria by adding IPTC and then purified by Ni2+ -NTA column. The purified protein was used as an immunogen to prepare polyclonal antibody ( pAb) of AD-004. The specificity of the antibody was detected by Western blotting. Immunohistochemical staining was performed in the mouse adrenal and testis via pAb of AD-004. Results Hisfused AD-004 was expressed efficiently in the prokaryotic system. Western blot analysis showed that the polyclonal antibody was duly bound to purified AD-004 with high specificity and sensitivity. AD-004 could be abundantly identified in the adrenal medulla and mainly expressed in the Leydig cells of testis. Conclusion The mouse protein of AD-004 is obtained from the prokaryotic expression system. The rabbit anti-AD-004 antibody has been prepared successfully. AD-004 protein is mainly localized in the interstitium of testis, suggesting that AD-004 may play a role in the synthesis of sex-steroid hormone.

Preparation and identification of polyclonal antibody against gastric cancer-related protein GCRG224 / 解放军医学杂志

Objective To prepare a polyclonal antibody against gastric cancer-related protein GCRG224.Methods The thioredoxin/GCRG224 fusion protein was expressed in E.coli.A polyclonal antibody against GCRG224 was obtained by immunizing a rabbit with the purified GCRG224 protein.The titer and specificity of the antibody were determined by ELISA and Western-blot,respectively.Results The thioredoxin/GCRG224 fusion protein with relative molecular mass of 16.8kD was over-expressed in E.coli.The purity of expressed products directly purified from a denaturing polyacrylamide gel was about 100%.The polyclonal antibody against GCRG224 was obtained.The ELISA titer of antiserum against GCRG224 was about 1∶256 000.Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically.Conclusion The polyclonal antibody against GCRG224 has been successfully prepared,which lays the foundation for further study on the biological function and the possible role of the GCRG224 in the development of gastric carcinoma.