Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a ‘wound healing’ assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.     

农业重组微生物生物安全研究进展

综述了近年来农业重组微生物的研究现状和进展 ,详细介绍农业重组微生物环境释放的监控方法以及对环境的冲击作用 ,对一些有关生物安全研究的热点问题进行了探讨     

Application of artificial neural networks to the analysis of two‐dimensional fluorescence spectra in recombinant E coli fermentation processes

A two‐dimensional (2D) spectrofluorometer was used to monitor various fermentation processes with recombinant E coli for the production of 5‐aminolevulinic acid (ALA). The whole fluorescence spectral data obtained during a process were analyzed using artificial neural networks, ie self‐organizing map (SOM) and feedforward backpropagation neural network (BPNN). The SOM‐based classification of the whole spectral data has made it possible to qualitatively associate some process parameters with the normalized weights and variances, and to select some useful combinations of excitation and emission wavelengths. Based on the classified fluorescence spectra a supervised BPNN algorithm was used to predict some of the process parameters. It was also shown that the BPNN models could elucidate some sections of the process's performance, eg forecasting the process's performance. Copyright © 2005 Society of Chemical Industry     

Effect of temperature on recombinant protein production using the Bm5/Bm5.NPV expression system

A series of experiments have been conducted using a recombinant baculovirus/insect cell expression system (Bm5/Bm5.NPV.CAT) to establish the optimum temperature for both cell growth and virus infection. Bm5 cell growth was found to be limited at temperatures below 22°C and ceased completely at temperatures above 34°C. In the range between 24 and 28°C, final cell densities always reached 96% of the highest achievable viable cell density. The shortest population doubling time was obtained at 28°C. Overall, a consistent increase in metabolism with increasing temperatures was observed. During the infection/viral replication phase, an increase in the temperature from 25 to 31°C resulted in a faster decrease in viable cell density and an earlier production of chloramphenicol acetyltransferase (CAT). Furthermore, protein yield at temperatures above 28°C was significantly reduced. Overall, the best temperature for the infection phase for the Bm5/Bm5.NPV expression system was found to be 25°C when the cells are cultured in serum free media.     

Ethanol production from raw starch by a recombinant yeast having saccharification and fermentation activities

In order to develop a method for converting raw starch into ethanol efficiently, direct fermentation of ozonized raw starch using a recombinant yeast was investigated. Ozonolysis was carried out as a pretreatment to convert raw starch into ethanol rapidly and efficiently, and then the effect of the ozone degradation conditions on the degree of polymerization and the amount of amylose in a raw starch was determined. Since the degree of polymerization was low and the amount of amylose was high, raw starch treated with an ozone concentration of 40 gm^?3 and an ozonation time of 30 min was the material chosen for alcohol fermentation. Though the recombinant yeast could not convert the untreated raw starch, it converted the soluble starch and the ozonized raw starch at a comparatively high yield into ethanol. About 56% of the ozonized raw starch decomposed, and the ethanol concentration obtained from the ozonized raw starch was markedly greater than that obtained from untreated raw starch. The dynamic behavior of cell growth, substrate degradation, and ethanol production was examined in a continuous culture under various dilution rates, and the optimal dilution rate, ie 0.15 h^?1, was determined for maximizing the ethanol productivity (amount of ethanol produced per unit time). © 2002 Society of Chemical Industry     

聚氨酯泡沫固定化重组枯草杆菌三段培养生产青霉素酰化酶

引　言青霉素G酰化酶 (penicillinGacylase ,EC3 5 1 11,PGA)是 β 内酰胺类抗生素工业中的关键酶之一 .目前主要采用大肠杆菌和巨大芽孢杆菌进行工业化生产 ,但由于产酶水平较低、变温发酵和需要苯乙酸诱导等缺点 ,国内外研究者尝试采用各种基因重组技术 ,以期大幅度提高产酶     

用KOH提取重组大肠杆菌中的PHA

用碱消化法从重组大肠杆菌中提取了PHA. 研究了碱液浓度、温度、消化时间及消化过程液固比等参数对产品PHA质量的影响. 提出了二段消化工艺,提高了PHA的产品纯度,所得PHA产品纯度为98.3%,同时较大幅度地降低了PHA的提取成本.     

枯草芽孢杆菌脂肪酶表达质粒的构建及其表达

以枯草芽孢杆菌168(B.subtilis 168)染色体为模板PCR扩增出P43启动子,与大肠杆菌-枯草杆菌穿梭质粒pUBC19相连得到表达载体pUBC-P43,然后将枯草芽孢杆菌脂肪酶基因lipA克隆到载体pUBC-P43启动子下游,得到重组质粒pUBCPL并转化B.subtilis TZ10.经中性红油脂平板、酶切和PCR方法鉴定得到重组菌TZ10/pUBCPL.宿主菌TZ10是B.subtilis DB104染色体缺失了lipA基因后获得.重组菌经初步发酵,以橄榄油为底物测定发酵上清液最高脂肪酶活力为49.1 U·L-1,而相应菌株DB104发酵最高酶活力仅为11.4 U·L-1.     

Construction, properties and specific fluorescent labeling of a bovine prothrombin mutant engineered with a free C-terminal cysteine

To define the role of phosphatidylserine-induced conformationalchanges in prothrombin activation during blood coagulation,a recombinant bovine prothrombin was constructed, characterizedand shown to have a globally native-like conformation. We introduceda cysteine to replace the penultimate residue (Gly581) of apreviously constructed active site mutant, and expressed thedouble mutant in Chinese hamster ovary cells at the level of0.6 µg/ml of cell culture medium. Specific labeling withfluorescein maleimide was accomplished by limited reductionwith dithiothreitol to free the engineered cysteine while maintainingthe native-like functional properties of the molecule. The averagestoichiometry of labeling was 0.84 probe/protein. The locationof the probe at the C-terminus was confirmed by proteolysisby native thrombin, by Taipan venom, and by carboxypeptidaseY. Both the double mutant and labeled prothrombin could be activatedby snake venoms and the prothrombinase but, as expected, thedouble mutant meizothrombin did not autolyze as does nativemeizothrombin. Thus, for the first time, a native-like but specificallylabeled prothrombin has been constructed. This molecule willbe an essential tool for elucidating the structural role ofmembranes during prothrombin activation. In addition, the methodsdescribed might be usefully applied to labeling of an odd, engineeredcysteine in other disulfide bond-containing proteins.