Improving the quality of a recombinant rabbit monoclonal antibody against PLXDC2 by optimizing transient expression conditions and purification method.

Rabbit monoclonal antibodies (mAbs) have many advantages over mouse antibodies in biological research and diagnostics applications because they exhibit high affinity and specificity. However, the methods of recombinant rabbit mAb production have not been optimized to the same extent as techniques used to produce mouse and human mAbs. In this study, we sought to optimize the production of a recombinant rabbit mAb against human plexin domain containing protein 2 (PLXDC2), a known cell surface antigen, by culturing HEK293-6E cells transfected with antibody-encoding genes at two different temperatures and by purifying the end-product by three different chromatography methods. The quality and function of purified antibody preparations were checked by electrophoresis and western blot analysis. The secreted rabbit mAb produced by a combination of culturing at 32 °C, purification by ammonium sulfate fractionation, and diethylaminoethyl resin (DEAE) ion exchange chromatography was of high quality. In contrast, the antibody produced by the cells grown at 37 °C for 6 days after transfection and purified by Protein A/G affinity method was low quality. Hypothermic conditions during production reduced protein heterogeneity probably by favorably affecting the levels of glycosylation and aggregation. In particular, according to western blotting data, CIMmultus DEAE chromatography that utilizes monolithic columns not only excluded inferior charge variants resulting from nonspecific reactions but also yielded rabbit mAb that was of better quality than commercially available rabbit polyclonal antibodies. The combination of techniques suggested by us may be a general approach to enhance product quality of rabbit mAbs produced by transient expression systems.

Combination cellulose plate (non-agar solid support) and agar plate method improves isolation of fungi from soil.

This is the first report describing the improved isolation of common filamentous fungi via a method combining cellulose plate and agar plate system. A cellulose plate is a porous plate made of nanofibrous crystaline cellulose. Isolating fungi from soils using these types of media separately resulted in the number of fungal colonies appearing on cellulose plates being lower than that on agar plates. However, the number of actual fungal species isolated using cellulose plates alone was more or less the same as that found using agar plates. Significantly, the diversity of isolates using a combination of the two media was greater than using each media individually. As a result, numerous new or rare fungal species with potential, including previously proposed new species, were isolated successfully in this way. All fungal colonies, including the Penicillium species, that appeared on the cellulose plate penetrated in potato dextrose were either white or yellow. Cultivation on cellulose plates with added copper ion overcomes the change in coloration, the colonies appearing as they do following cultivation on potato dextrose agar.

Screening of optimal cellulases from symbiotic protists of termites through expression in the secretory pathway of Saccharomyces cerevisiae.

For direct and efficient ethanol production from cellulosic materials, we screened optimal cellulases from symbiotic protists of termites through heterologous expression with Saccharomyces cerevisiae. 11 cellulases, belonging to glycoside hydrolase families 5, 7, and 45 endoglucanases (EGs), were confirmed to produce with S. cerevisiae for the first time. A recombinant yeast expressing SM2042B24 EG I was more efficient at degrading carboxylmethyl cellulose than was Trichoderma reesei EG I, a major EG with high cellulolytic activity.

Promotion of efficient Saccharification of crystalline cellulose by Aspergillus fumigatus Swo1.

Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.

Phylogenetic analysis of cellulolytic enzyme genes from representative lineages of termites and a related cockroach.

The relationship between xylophagous termites and the protists resident in their hindguts is a textbook example of symbiosis. The essential steps of lignocellulose degradation handled by these protists allow the host termites to thrive on a wood diet. There has never been a comprehensive analysis of lignocellulose degradation by protists, however, as it has proven difficult to establish these symbionts in pure culture. The trends in lignocellulose degradation during the evolution of the host lineage are also largely unknown. To clarify these points without any cultivation technique, we performed meta-expressed sequence tag (EST) analysis of cDNA libraries originating from symbiotic protistan communities in four termite species and a wood-feeding cockroach. Our results reveal the establishment of a degradation system with multiple enzymes at the ancestral stage of termite-protistan symbiosis, especially GHF5 and 7. According to our phylogenetic analyses, the enzymes comprising the protistan lignocellulose degradation system are coded not only by genes innate to the protists, but also genes acquired by the protists via lateral transfer from bacteria. This gives us a fresh perspective from which to understand the evolutionary dynamics of symbiosis.

Heterologous expression and characterization of an endoglucanase from a symbiotic protist of the lower termite, Reticulitermes speratus.

RsSymEG, an endoglucanase of glycosyl hydrolase family (GHF) 7 encoded by a transcript isolated from the symbiotic protist of the termite Reticulitermes speratus, is expressed in Aspergillus oryzae. Interestingly, purified RsSymEG1 has a relatively higher specific activity (603 micromol min(-1) mg(-1) protein) and V(max) value (769.6 unit/mg protein) than previously reported data for GHF7 endoglucanase of Trichoderma ressei. It also has the same K(m) value (1.97 mg/ml) with Clostridium cellulolyticum enzymes that contain cellulose binding module, a property indicative of high affinity to substrate, though no cellulose binding module is found within it. Thin-layer chromatography analysis revealed that RsSymEG1 preferentially hydrolyzes the beta-1,4-cellulosic linkage of cellodextrins into cellobiose and glucose.

Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus.

To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.