Reconstitution of (1→3)-ß-D-glucans measurement system using recombinant Limulus polyphemus Factor G.

Horseshoe crab Factor G is a heterodimeric serine protease zymogen that is activated by (1→3)-ß-D-glucans (BDG) from fungal cell walls. This reaction is used in diagnostic agents for deep-seated mycosis. At present, functional analysis using Factor G from Tachypleus tridentatus has been performed, and genetic information has been published, but reconstitution using recombinant proteins has not yet been achieved. In this study, we cloned the genes for Factor G α and ß from Limulus polyphemus; two gene sequences were obtained for Factor G α and seven for ß. The obtained L. polyphemus Factor G α was used to specifically remove BDG from the culture medium for eliminating the activator BDG. The optimal combination for each sequence was examined with BDG removal medium, and a combination was found that featured BDG-dependent activity. These results indicate that a BDG assay system using recombinant Factor G is feasible in reconstitution. This research will support future reagent development that does not require natural horseshoe crab resources. KEY POINTS: • Cloned novel Factor G α subunit and ß subunit genes from L. polyphemus • Proposed a method of removing BDG without reducing culture medium performance • Identified combination of recombinant α and ß subunits for BDG-dependent activation.

Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells.

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here, we demonstrate a new method to see endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps: First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG- and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously see the dynamics of the two loci in single living cells. This shows a close association between the two loci in the majority of cells, but the loci markedly separate from the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof of principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.

Dimerization of bisphenol A by hyper lignin-degrading fungus Phanerochaete sordida YK-624 under ligninolytic condition.

Bisphenol A (BPA) was treated with hyper lignin-degrading fungus Phanerochaete sordida YK-624 under ligninolytic condition. After preculturing P. sordida YK-624 for 4 days, BPA (final concentration, 1 and 0.1 mM) was added to cultures. Both 1- and 0.1-mM BPA were effectively decreased within a 24-h treatment and two metabolites were detected. Two metabolites (5,5'-bis-[1-(4-hydroxy-phenyl)1-methyl-ethyl]-biphenyl-2,2'-diol and 4-(2-(4-hydroxy-phenyl) propan-2-yl)-2-(4-(2-(4-hydroxyphenyl) propan-2-yl) phenoxy)phenol) were identified by ESI-MS and NMR analysis. These results indicated that BPA was oxidized to BPA phenoxy radicals by ligninolytic enzymes and then dimerized at extracellular region.

Mamestra brassicae NIAS-Mb-32 cell strain 2g2 enables high-yield recombinant protein production in baculovirus-free and baculovirus-based insect cell expression.

The production of recombinant proteins using insect cells has been widely used for over 30 years, which contributing to life science research and biotechnology. Insect cells exhibiting enhanced N-glycosylation and recombinant protein productivity enhance the productivity of the baculovirus-insect cell system (BICS). A new highly proliferative insect cell strain, 2g2, was established from the Mamestra brassicae pupa ovary cell strain NIAS-MB-32 (RCB0413) to address the problem of Sf-rhabdovirus and to explore the newly available possibilities in BICS as well as Sf9, such as increased protein production and recombinant baculovirus amplification. The high-growth cell strain 2g2 was examined for its recombinant protein production ability and baculovirus productivity; moreover, the activity of the produced recombinant proteins was examined using Sf9 as a benchmark. Recombinant protein productivity and virus production by BICS in 2g2 was confirmed as equivalent to that of Sf9. Furthermore, we produced the severe acute respiratory syndrome coronavirus 2 spike protein in a baculovirus-free system and compared its productivity, binding activity with human angiotensin-converting enzyme 2, and N-glycosylation. The productivity and bioactivity were found to be equal to or better than that of Sf9. Moreover, N-glycosylation analysis revealed that the glycans derived from the 2g2-produced glycoproteins were mostly of the high mannose type as Sf9. Therefore, 2g2 may have the same N-glycosylation ability as Sf9. Finally, the Sf-rhabdovirus was confirmed to be negative in 2g2. Our results demonstrated that the novel insect cell strain 2g2 can serve as a protein production tool in scientific research and industrial biotechnology.

Hydroxylation of bisphenol A by hyper lignin-degrading fungus Phanerochaete sordida YK-624 under non-ligninolytic condition.

Bisphenol A (BPA) is one of the representative compounds of the endocrine disrupting compounds group and the highest volume chemicals produced worldwide. As a result, BPA is often detected in many soil and water environments. In this study, we demonstrated the transformation of BPA from liquid cultures inoculated with hyper lignin-degrading fungus Phanerochaete sordida YK-624. Under non-ligninolytic condition, approximately 80% of BPA was eliminated after 7d of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of a metabolite isolated from the culture supernatant suggested that BPA was metabolized to hydroxy-BPA, 4-(2-(4-hydroxyphenyl)propan-2-yl)benzene-1,2-diol, which has a much lower estrogenic activity than BPA. In addition, we investigated the effect of the cytochrome P450 inhibitor piperonyl butoxide (PB) on the hydroxylation of BPA, markedly lower transformation activity of BPA was observed in cultures containing PB. These results suggest that cytochrome P450 plays an important role in the hydroxylation of BPA by P. sordida YK-624 under non-ligninolytic condition.