Sensitive and specific quantification of antisense oligonucleotides using probe alteration-linked self-assembly reaction technology.

Quantitative bioanalysis is essential when establishing pharmacokinetic properties during the drug development process. To overcome challenges of sensitivity, specificity and process complexity associated with the conventional analysis of antisense oligonucleotides (ASOs), a new approach to nonenzymatic hybridization assays using probe alteration-linked self-assembly reaction (PALSAR) technology as a signal amplifier was evaluated. PALSAR quantification of ASOs in mouse tissue and plasma was able to achieve a high sensitivity ranging from 1.5 to 6 pg/ml, intra-/interday accuracies in the range of 86.8-119.1% and 88.1-113.1%, respectively, and precision of ≤17.2%. Furthermore, crossreactivity of 3'n-1, a metabolite with a single base difference, was <1%. Our approach provides an auspicious method for distinguishing metabolites and detecting ASOs with high sensitivity and specificity.

Feasibility of using PALSAR technology as a signal amplifier for antibody bridging assay.

The immunogenicity testing of oligonucleotide drugs using an antibody bridging assay has been scarcely investigated. We developed a highly sensitive antibody bridging assay model and assessed it using probe alteration link self-assembly reactions (PALSAR) technology as a signal amplifier. Methods: The concentration of each probe was optimized, and the bridging assay model was compared with and without signal amplification. Cut-point and analytical sensitivity were determined, and accuracy, precision and drug tolerance were evaluated. Results: The PALSAR bridging assay achieved a net signal 21-36 times higher than that obtained with the conventional method. The analytical sensitivity achieved was 48.8 ng/ml, with adequate accuracy, precision and drug tolerance. Conclusion: PALSAR technology is feasible for developing an antibody bridging assay using oligonucleotides as capture and detection probes.

Bioconversion of substituted naphthalenes and ß-eudesmol with the cytochrome P450 BM3 variant F87V.

Bioconversion of various substituted naphthalenes that contain 1-methoxy- and 1-ethoxy-naphthalenes, methylnaphthalenes, dimethylnaphthalenes, and naphthalenecarboxylic acid methyl esters were performed using recombinant Escherichia coli cells, which expressed the gene coding for a cytochrome P450 BM3 variant F87V (P450 BM3 (F87V)) that was N-terminally fused to an archaeal peptidyl-prolyl cis-trans isomerase. In addition, bioconversion experiments with the same substrates were carried out using those that expressed the phnA1A2A3A4 genes for a polycyclic aromatic hydrocarbon (PAH)-dihydroxylating dioxygenase, which originated from a PAH-utilizing marine bacterium Cycloclasticus sp. strain A5. Consequently, a variety of mono-hydroxylated derivatives were generated from these substituted naphthalenes. Oxidative aryl coupling was found to produce a novel compound 4,4'-diethoxy-[2,2']-binaphthalenyl-1,1'-diol from 1-ethoxynaphthalene with the E. coli cells expressing the P450 BM3 (F87V) gene. This recombinant E. coli was further shown to introduce the hydroxyl group regio- and stereo-specifically into a sesquiterpene ß-eudesmol.

An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool.

The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin alpha-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3' end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21-25 kDa) toxic to host cells or two antibody fragments (25-36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities.

Alkali- and halo-tolerant catalase from Halomonas sp. SK1: overexpression in Escherichia coli, purification, characterization, and genetic modification.

A catalase gene, ohktA, from an alkali- and halo-tolerant bacterium, Halomonas sp. SK1, on the pKK223-3, was expressed in the catalase-lacking Escherichia coli strain UM2. Highly purified catalase showing a single band on SDS-PAGE was obtained by two liquid chromatography steps on DEAE-Toyopear1 and Chelating-Sepharose Fast Flow. The enzyme, oHktA, shows high catalase activity with a pH optimum at 10, and the activity was stable in 4 M KC1. This enzyme is thermo-sensitive, showing a significant loss of activity within 5 minutes at 37 degrees C. To modify the stability of the catalase, the addition of domain II of the heat stable Mn catalase from Thermus thermophilus to the C-terminus was made. When coexpressed with a chaperone (PhFKBP29) gene product, peptidyl-prolyl cis-trans isomerase, from a thermophilic bacterium, a chimeric catalase was produced in the soluble fraction. The stability of this catalase in the range of 37 degrees -45 degrees C was improved and it was stable for more than 1 h at 37 degrees C.

Expression of long- and short-type FK506 binding proteins in hyperthermophilic archaea.

It has been reported that the hyperthermophilic archaeon, Methanococcus jannaschii, possesses two FKBP (FK506 binding protein) genes in the genome, one being 26 kDa FKBP (long-type FKBP) and the other, 18 kDa FKBP (short-type FKBP). FKBP is a family of peptidyl-prolyl cis-trans isomerases (PPIases). In order to clarify the difference between their roles in archaeal cells, they were expressed in Escherichia coli, and their PPIase and chaperone-like protein-folding activities were investigated. The catalytic efficiency of the PPIase activity of the long-type FKBP was significantly lower than that of short-type FKBP (less than 1/1000) which is comparable to that of human FKBP12. Both FKBPs showed chaperone-like protein-folding activity to enhance the refolding yield of an unfolded protein (Thermoplasma citrate synthase) in vitro. The chaperone-like protein-folding activity of the short type was higher than that of the long type. While the intracellular content of long-type FKBP in M. jannaschii tended to increase, that of short-type FKBP obviously decreased at growth temperatures higher than the optimum of 85 degrees C. In Pyrococcus horikoshii, another hyperthermophilic archaeon, the intracellular content of long-type FKBP did not change with temperature (80-102 degrees C). These results suggest that long-type FKBP functions at any temperature in the cells as a chaperone to maintain the folding states of intracellular proteins. On the other hand, short-type FKBP may be required at lower temperatures. Peptidyl-prolyl cis-trans isomerization is known to be a rate-limiting step in protein-folding and is slower at low temperature. Since the PPIase activity of short-type FKBP was much stronger than that of the long type, it may be required to accelerate the folding of intracellular proteins and for the hyperthermophilic cell to live at low growth temperatures.

Two kinds of archaeal group II chaperonin subunits with different thermostability in Thermococcus strain KS-1.

The thermostability of the recombinant alpha- and beta-subunit homo-oligomers (alpha16mer and beta16mer) and of natural chaperonins purified from cultured Thermococcus strain KS-1 cells was measured to understand the mechanism for the thermal acclimatization of T. KS-1. The beta-subunit content of the natural chaperonin from cells grown at 90 degrees C was higher than that at 80 degrees C. The optimum temperature for ATPase activity of the natural chaperonins was 80-90 degrees C, whereas that for alpha16mer and beta16mer was 60 degrees C and over 90 degrees C respectively. Judging from the ATPase activity, beta16mer was more thermostable than alpha16mer. The thermostabilities of the natural chaperonins were intermediate between alpha16mer and beta16mer, whereas the natural chaperonin with a higher beta-subunit content was more stable than that with a lower beta-subunit content. Native polyacrylamide gel electrophoresis (PAGE) revealed that the chaperonin oligomers thermally dissociated to their ATPase-inactive monomers. The thermal denaturation process monitored by circular dichroism showed that the free beta-subunit was more stable than the free alpha-subunit, and that the secondary structure of the chaperonin monomer in the oligomer was more stable than that in the free monomer. These results suggest that the structure of these subunits was stabilized in the oligomer, and that an increase in the beta-subunit content conferred higher thermostability to the natural hetero-oligomeric chaperonin.

FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli.

The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.