The gene HCAP1 (HCC-associated Protein 1), one variant of GEMIN4, has been mapped in a minimum LOH region on chromosome 17p13.3 and encodes a 1047-amino acid protein. Function predictions based on the amino acid sequence of protein HCAP1 revealed it to contain one helix-loop-helix motif and one leucine zipper domain. Using yeast two-hybrid screening, five zinc-finger proteins were identified as HCAP1-interacting proteins. Among them, NDP52 (nuclear dot protein 52) appeared most frequently in positive clones and was the most strongly interacting protein. Then, the interaction between HCAP1 and NDP52 was confirmed by GST pull-down assay and a coimmunoprecipitation experiment. Moreover, an immunofluorescent staining assay indicated that NDP52 colocalizes with HCAP1 in the cytoplasm. By deletion analysis, the leucine zipper domain of HCAP1 and the zinc finger domain of NDP52 were identified as important regions responsible for the interaction.

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Tα1 linked by different lengths of (G4S)n (n=1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n=1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1 (n=1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex 75 gel filtration and analyzed by SDSPAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4S)n-Tα1 (n=1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n=1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex™ 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1 (n=1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.

CDC16Hs是细胞周期末期促进复合物（APC）的亚基。利用基于LexA的酵母双杂交系统，把它作为诱饵蛋白筛选人胎脑文库，发现它与DNA双链断端修复蛋白Ku80的羧基端相互作用。CDC16Hs和全长Ku80的结合通过pull down实验在体外得到验证。

将编码人的血管生成素样蛋白4（ANGPTL4）的cDNA克隆至分泌表达载体pPIC9k上，在Pichia Pas-toris酵母宿主菌SMD1168中获得分泌表达；用MTT方法检测的细胞生长抑制实验结果表明，发酵液中分泌表达的重组蛋白ANGPTL4对人脐静脉内皮细胞的生长具有一定的抑制作用。

化学合成人纤溶蛋白酶原K5（pK5）的编码基因并克隆到毕赤氏酵母表达系统的分泌型载体pPIC9K上，将重组质粒经BglⅡ单酶切后电转化Pichia pastoris GS115菌株，筛选出对G418有高抗性和在MM培养基上生长缓慢的转化子。经摇瓶发酵和甲醇诱导后，用15%SDS-PAGE检测发酵上清液，表明有重组蛋白pK5的高表达。经CM.Sepherose离子交换柱和Superdex 75分子筛层析两步分离纯化，获得了纯度达到98%的rpK5。用MTT方法检测的结果表明，纯化的rpK5可显著地抑制人血管内皮细胞的生长。