Effect of Ca2+ mobilization on release and activation of matrix metalloproteinases in hepatocellular carcinoma cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effect of Ca(2+) mobilization on release and activation of matrix metalloproteinases (MMPs) in human hepatocellular carcinoma cells.</p><p><b>METHODS</b>Ca(2+) and chemicals which can induce or inhibit Ca(2+) mobilization were added into human SMMC-7721 hepatoma cells in vitro. SDS-PAGE protein electrophoresis and gelatin zymography analysis were carried out to detect the changes of release and activation of MMPs in the cell culture supernatant.</p><p><b>RESULTS</b>Addition of CaCl(2) into culture system resulted in an enhanced secretion and activation of MMP-2 and MMP-9 in a dose-dependent manner. At a dose of 0.8 mmol/L CaCl(2), it maintained a stable high level of MMPs, especially of MMP-2 with (109.71 +/- 27.93)% elevation as compared to the cells without CaCl(2) addition (P < 0.001). SDS-PAGE analysis showed that most secreted proteins were MMPs (MMP-2 and MMP-9) when the cells cultured in media without serum. Thapsigargin (Tg, 4 micromol/L), an inducer of intracellular Ca(2+) stores depletion, significantly enhanced the release and activation of MMP-2 and MMP-9, compared to the control with (58.63 +/- 31.04)% elevation (P < 0.05), while the inducing effect of Tg on MMPs release and activation was significantly inhibited by S-nitro-N-acetylpenicillamine (SNAP, 200 micromol/L), an NO donor.</p><p><b>CONCLUSION</b>Intracellular Ca(2+) regulation pathways may play an important role in the process of release and activation of MMPs.</p>

Non-fused expression of HAb18GEF by reducing stability of translational initiation region in mRNA / 生物工程学报

To express the extracellular fragement of hepatoma associated antigen HAbl8G(HAb18GEF) in E. coli efficiently in a non-fusing way, the cDNA of HAb18GEF gene was inserted into prokaryotic expression vector pET21a + . The secondary structure and codon adaptation of translational initiation region (TIR, from-30 to + 39) in mRNA of recombinant vector HAb18GEF/ pET21a + was predicted simultaneously by computer-aided design. Stable Stem-Loop structures and many low-usage codons were detected in mRNA-TIR of non-optimized recombinant HAb18GEF/pET21a + vector. The stability of mRNA-TIR in recombinant HAb18GEF/pET21a + vector was reduced with following methods: (1) optimization of secondary structure (2) optimization of codon adaptation. These optimization were realized by non-continual site-directed mutagenesis without changing any amino acid sequence in TIR. After being checked through restriction endonuclease digestion and confirmed through nucleotide sequencing, the pre-optimized and post-optimized recombinant vectors were transformed into competent E. coli JM109-DE3. The resulted recombinant clones were selected randomly and induced by IPTG at 37 degrees C. The induced production of these recombinants was analyzed by SDS-PAGE, indirect ELISA, Western blot, and cell fractionation assay. The amount of HAb18GEF mRNA was also detected by RNA dot blot between pre-optimized recombinant and post-optimized recombinant. The results revealed that recombinant non-fused vectors HAb18GEF/pET21a + were successfully constructed and optimized in the secondary structure and codon adaptation of TIR respectively. The HAb18GEF was expressed efficiently in a non-fusing way in recombinant E. coli by secondary structure optimization or codon adaptation optimization. Whereas, no expression of HAb18GEF was detected in pre-optimized recombinants. The non-fused expression products-HAb18GEF, mainly as inclusion body in E. coli, yielded highly above 29.3%. A trait of expression HAb18GEF was also detected both in intermembrane space and in culture medium due to over-expression and cell leakage. Difference in non-fused expression level of HAb18GEF between secondary structure optimization and codon adaptation optimization was negligible. No difference in amount of transcribed mRNA of HAb18GEF between the pre-optimized and the post-optimized recombinants was detected. To sum up, it's feasible to express hepatoma associated antigen HAb18GEF in a non-fusing way by reducing the stability of TIR in mRNA.

Recovery and purification of monoclonal antibody producted by continuous perfusion culture using expended bed adsorption / 生物工程学报

Monoclonal antibody producted by continuous perfusion culture was recovered and purified by expended bed adsorption chromatography. A packed bed column XK16/20 was used for method scouting with Streamline SP adsorbent. Two expended bed columns Streamline-25 and -50 were used for method optimization and pilot scale experiment, respectively. The recovery yield of monoclonal antibody was above 90% in a 5 - 7 fold enhanced purity and 10 fold increased concentration. According to the different concentration of monoclonal antibody in cell culture broth, about 18 - 50L fluid can be treated in a single cycle. MAb purification from lab scale (400mg per cycle) to a small pilot scale (2g per cycle) has been achieved. Compared with packed bed adsorption, the preparation cycle was half shortened, and the cost of production and the complexity of process were decreased markedly. It has been proven that a purification process based on expended bed adsorption technique is simple, efficient and economical.