Genomic and exoproteomic diversity in plant biomass degradation approaches among Aspergilli.

We classified the genes encoding carbohydrate-active enzymes (CAZymes) in 17 sequenced genomes representing 16 evolutionarily diverse Aspergillus species. We performed a phylogenetic analysis of the encoding enzymes, along with experimentally characterized CAZymes, to assign molecular function to the Aspergilli CAZyme families and subfamilies. Genome content analysis revealed that the numbers of CAZy genes per CAZy family related to plant biomass degradation follow closely the taxonomic distance between the species. On the other hand, growth analysis showed almost no correlation between the number of CAZyme genes and the efficiency in polysaccharide utilization. The exception is A. clavatus where a reduced number of pectinolytic enzymes can be correlated with poor growth on pectin. To gain detailed information on the enzymes used by Aspergilli to breakdown complex biomass, we conducted exoproteome analysis by mass spectrometry. These results showed that Aspergilli produce many different enzymes mixtures in the presence of sugar beet pulp and wheat bran. Despite the diverse enzyme mixtures produced, species of section Nigri, A. aculeatus, A. nidulans and A. terreus, produce mixtures of enzymes with activities that are capable of digesting all the major polysaccharides in the available substrates, suggesting that they are capable of degrading all the polysaccharides present simultaneously. For the other Aspergilli, typically the enzymes produced are targeted to a subset of polysaccharides present, suggesting that they can digest only a subset of polysaccharides at a given time.

Efficacy of an insulin-like growth factor-interleukin-3 fusion protein in reversing the hematopoietic toxicity associated with azidothymidine in mice.

The effect of 406, a novel fusion protein between the N-terminal sequence of the insect insulin-like peptide, bombyxin, human insulin-like growth factor II and mouse interleukin 3 was investigated in its capacity to abrogate the toxic effects of azidothymidine (AZT) in C57BL/6 mice. Mice receiving 2.5 mg/ml AZT in their drinking water were concurrently treated with daily s.c. injections of 14, 140 or 1400 ng 406 for 4 wk. AZT-treated mice had a lower total weight, hemoglobin content and white blood cells than non treated controls. 406 significantly increased the number of circulating white blood cells at all doses, and the optimal effects were observed at a dose of 140 ng/mouse. Using this optimal dose, 406 completely abrogated the AZT-mediated weight loss. The effects on erythroid cells depended on the severity of the AZT-induced anemia. The amounts of hemoglobin were equal or slightly lower than those of controls under conditions of mild anemia, but were significantly higher than controls under conditions of severe anemia. 406 significantly increased the number of all hematopoietic colony-forming cells in bone marrow and spleen, but the effects were particularly striking in granulocyte-macrophage precursors. Blood glucose levels did not change at optimal or suboptimal 406 doses but increased at a dose of 1.4 microg/mouse. These experiments demonstrate the usefulness of these IGF-cytokine fusion proteins, whose low cost production represents a significant advantage for future in vivo studies.

Preparation of a recombinant chimaera of insulin-like growth factor II and interleukin 3 with high proliferative potency for haemopoietic cells.

We have found that a slightly modified insulin-like growth factor II (IGF II) consisting of a chimaera of bombyxin and human IGF II (BOMIGF) is properly secreted in insect cells by using the baculovirus expression system. Human interleukin 3 (IL-3) was attached to the C-terminal amino acid residue of BOMIGF with peptide linkers containing five or twelve residues. Only the chimaera with the 12-residue linker had biological activities of both IGF II and IL-3. The chimaera had a significantly higher mitogenic activity than IL-3 in cell cultures of the human haemopoietic cell line TF-1 and its effect could be observed even at femtomolar concentrations. It was also able to stimulate thymidine incorporation in IGF II-dependent bovine fetal erythroid cells. The chimaera significantly increased the number of macroscopic haemopoietic colonies in cultures of human peripheral blood in comparison with IL-3 or mixtures of IL-3 and BOMIGF in vitro. Subcutaneous injection of a BOMIGF-mouse IL-3 chimaera in normal C57BL/6 mice resulted in a significant increase of the number of spleen stem cells producing macroscopic haemopoietic colonies. This new system for the biosynthesis of IGF-cytokine fusion proteins in insect cells might prove advantageous for the low-cost and high-yield production of molecules with complementary or synergistic biological activities.

The influence of various insect cell lines, p10 and polyhedrin promoters in the production of secreted insulin-like growth factor-interleukin-3 chimeras in the baculovirus expression system.

A technique for the optimal synthesis of secreted fusion proteins between insulin-like growth factors (IGFs) and cytokines is described. The cDNA of BOMIGF, a fusion protein between the insect insulin-like peptide bombyxin and IGF II, has been linked to the gene of interleukin-3. The BOMIGF-interleukin 3 fusion gene was cloned downstream of the promoter regions of the p10 and polyhedrin proteins within baculovirus transfer vectors. A third, dual transfer vector was constructed with the gene inserted simultaneously behind p10 and polyhedrin promoters. Two different lepidopteran cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (BTI-TN-5B1-4) were infected with the recombinant baculoviruses obtained from the three transfer vectors. Trichoplusia ni cells produced the largest amount of recombinant protein. Although the efficiency of the three recombinant viruses was remarkably similar, the baculovirus with the gene present behind both promoters produced relatively more recombinant protein in host cells than those viruses driven with the polyhedrin or p10 promoters alone. The BOMIGF-interleukin-3 chimera was stable and continuously increased in the culture medium up to 5-6 days postinfection. Therefore the addition of a protease inhibitor was useful only at the stage of massive host cell death. Medium supplemented with copper sulfate was detrimental for the long-term production of the fusion protein.