Cellular internalization and trafficking of 20 KDa human growth hormone.

Twenty kilodalton human growth hormone (20K-GH) is the second most abundant GH isoform after the twenty-two kilodalton human growth hormone (22 K-GH) isoform. 20K-GH exhibits similar but not identical physiological activities as that of 22K-GH. The cell behaviour of 22K-GH has been extensively studied, but little or no information has been reported regarding 20K-GH. Here, we focussed on the internalization of 20K-GH. We found that the internalization of 20K-GH is rapid and occurs in a time- and dose-dependent manner. 20K-GH internalization is mediated by GHR. It appears that the internalization of 20K-GH and GHR into the cytoplasm is mediated by clathrin and/or caveolin. The current study indicates that 20K-GH can internalize into the cytoplasm and suggests that the internalized 20K-GH may exhibit different functions from those of 22K-GH.

An extract of Perilla stem inhibits Src homology phosphatase-1 (SHP)-1 and influences insulin signaling.

Protein tyrosine phosphatases (PTPs) are enzymes that catalyze protein tyrosine dephosphorylation of which Src homology phosphatase-1 (SHP-1) is one of the best-validated, a widely distributed intracellular tyrosine phosphatase that contains two SH2 domains. Down regulation of SHP-1 tyrosine phosphatases was significantly increased sensitivity to insulin in insulin signaling pathway. Through in vitro enzymatic reaction kinetics experiment, we found that the extract of Perilla stem was a potential inhibitor to δSHP-1, the catalytic domain of SHP-1 protein tyrosine phosphatase, and its IC(50) was 4ug/ml, and was more sensitive towards SHP-1than other PTPs, which indicated that SHP-1 might be a target of the extract of Perilla stem. It can strengthened the level of tyrosine phosphorylation of insulin receptor (IR) and extracellular signal-regulated protein kinase (ERK) in HepG2 cells, and then activated the insulin signaling pathway through inhibiting the protein phosphorylation of SHP-1. These results demonstrated that the extract of Perilla stem could play an important role for diabetes treatment through inhibiting the level of SHP-1 in insulin signaling pathway.

Expression and bioactive characterization of bacteriophage lysin gene of Bacillus anthracis in Escherichia coli / 生物工程学报

The lysin gene of Bacillus anthracis-diagnosing bacteriophage, obtained by PCR amplification,was cloned into the Escherichia coli exepression vector pET22b which has been cleaved by EcoR I and Nde I. The recombinant vector pET22b-gamma lysin was verified to be correctly constructed by PCR, sequencing and enzyme digestion, and highly expressed in E. coli BL21 (DE3), which accounted for about 40 percent of total protein in E. coli BL21 (DE3), while in the 5L fermentor the expression level reached 15g/L. After expression, disruption and purification with three-step chromatography, Streamline SP, SP HP and Sephacryl S-100, the recombinant gamma lysin was finally obtained with purity of higher than 95 percent as determined by gel scan. The final yield following SP HP was 19.1 percent, with a greater-than-350-fold increase in specific activity. The pure enzyme has been shown active to Bacillus anthracis, and not to E. coli, Bacillus subtilis and Bacillus cereus. Its specific activity was about 1400 u/mg.

Construction of phage display antibody library to MCF-7 cells and screening of single-chain antibodies against breast cancer cells / 生物工程学报

The aim of this study is to construct a phage display single-chain variable fragment (scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the library. The BALB/C mice were immunized with MCF-7 cells. Total RNA of spleens was isolated. The heavy-chain (VH) and light-chain variable region genes (VL) of the antibodies were amplified by RT-PCR and joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E) and the recombinant phagemids were used to transform competent E. coli TG1. The transformed TG1 cells were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The scFv fusion proteins were displayed on the surfaces of the recombinant phages. A phage display antibody library of repertoire of 1.2 x 10(6) clones was constructed. The specific antibodies against MCF-7 cells were enriched by 75 times after five rounds of affinity selection. Ten recombinant phages clones that exhibited specific binding to MCF-7 cells were identified. The specificity of those phage clones was analyzed by reactivity against HepG2 cells and Hela cells by ELISA. One of the selected phage clones against MCF-7cells was used to infect E. coli TOP10 to produce the soluble scFv antibodies after induction with IPTG. The strategy of construction and screening of antibody library directed against the whole tumor cells described in this report should be generally applicable to generate tumor cell-specific antibodies.