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.

利用同源重组的方法，构建了具有高拷贝数和高稳定性的新型酿酒酵母表达质粒。对酵母附加体型载体pHC11进行一系列改造，得到质粒pHC11R，并用适当的酶切线性化；利用PCR反应得到人IFNa2b表达单元片段，它的两端和线性化的pHC11R的两端具有50bp左右的同源区段。上述两个片段共转化酿酒酵母，在酵母体内经同源重组后得到表达质粒pHC11R-IFNa2b，该表达质粒与pHC11-IFNoa2b相比去除了质粒上用于在大肠杆菌中复制和筛选所需的序列，在宿主菌中的稳定性和拷贝数均有明显提高，同时外源基因的表达量也获得了一定程度的提高。在此基础上，进一步构建了带有不同表达元件的pHR系列载体，使得外源基因能够通过同源重组在酿酒酵母中快速、方便地克隆和表达。

The human novel gene pp5644 (GeneBank Accession No. AF289559) coding for 124 amino acids was recently cloned. Overexpression of pp5644 in Hela cells significantly inhibited the growth and colony formation. The pp5644-interacting protein FAPP1 (phosphatidylinositol-four-phosphate adaptor protein1) associated protein-1, called FASP1, was obtained by using yeast two-hybrid system. The interaction between pp5644 and FASP1 was experimentally confirmed by GST pull-down assay in vitro and co-immunoprecipitation assay in vivo. Co-localization of pp5644 and FASP1 in cytoplasm in Hela cells could further support the interaction. Based on the experimental results, it is suggested that pp5644 physically bind to FASP1 and the biological significance of this kind of interaction in vivo is discussed.

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.

Bcl-2/adenovirus E1B 19 kDa interacting protein 2-like, BNIP-2-like (BNIPL) is a recently cloned and characterized apoptosis-associated protein that shares 72% homology with BNIP-2. It is highly expressed in human placenta and lung. A yeast two-hybrid system was used to obtain two BNIPL-interacting proteins, MIF (macrophage migration inhibitory factor) and GFER (growth factor erv1 (Saccharomyces cerevisiae)-like). The interactions were con