Trans-splicing as a novel method to rapidly produce antibody fusion proteins.

To cultivate the use of trans-splicing as a novel means to rapidly express various antibody fusion proteins, we tried to express antibody-reporter enzyme fusions in a COS-1 co-transfection model. When a vector designed to induce trans-splicing with IgH pre-mRNA was co-transfected with a vector encoding the mouse IgM locus, the expression of V(H)-secreted human placental alkaline phosphatase (SEAP) as well as Fab-SEAP were successfully expressed both in mRNA and protein levels. Especially, the vectors encoding complementary sequence to Smu as a binding domain was accurate and efficient, producing trans-spliced mRNA of up to 2% of cis-spliced one. Since Smu sequence should exist in every IgH pre-mRNA, our finding will lead to the rapid production and analysis of various antibody-enzyme fusions suitable for enzyme-linked immunosorbent assay (ELISA) or antibody-dependent enzyme prodrug therapy (ADEPT).

Refolding of lactate dehydrogenase by zeolite beta.

We used zeolite beta as an adsorbing matrix to refold recombinant lactate dehydrogenase (LDH) protein collected as an insoluble aggregate from a bacterial expression system. The adsorption isotherm revealed that 1 g of zeolite adsorbed 200 mg of denatured LDH solubilized with a buffer containing 6 M of guanidine hydrochloride. The pH of the buffer had little effect on the adsorption, but this property was abolished by preincubation of the zeolite with polyethylene glycol (PEG) in a weight ratio of 1:10. These data suggest that the adsorption of LDH depends on the hydrophobicity of the zeolite surface, and that the adsorption of PEG to zeolite is sufficient to release LDH from its surface. LDH was thus released by refolding buffer containing PEG and arginine, and soluble LDH was obtained in its active enzymatic form. The addition of arginine dramatically increased the yield of LDH in a dose-dependent manner. The overall refolding efficiency was optimized to 35%.

Use of zeolite to refold a disulfide-bonded protein.

Zeolites are microporous crystalline aluminosilicates with a highly ordered structure. Using zeolite beta as an adsorbent, denatured/reduced hen egg lysozyme was refolded to the active form at high concentrations. The denatured/reduced lysozyme was adsorbed onto the zeolite and the protein was refolded by desorbing it into refolding buffer, consisting of redox reagents, guanidine hydrochloride, polyethylene glycol, and L-arginine. This zeolite refolding method could be highly effective for various kinds of proteins, refolding them with high efficiency even when they contain disulfide bonds.

Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surfaces in organic solvents: potential as a whole-cell biocatalyst in organic solvents.

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with alpha-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 x 10(4)-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 x 10(4)-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.

Construction of a selective cleavage system for a protein displayed on the cell surface of yeast.

We constructed a novel protein-purification system in which Saccharomyces cerevisiae with a protein displayed on the cell surface is harvested and the displayed protein is then cleaved from the cell surface. GFPuv was used as a model protein in this cell surface engineering experiment. In this system, the C-terminal 320 amino acids of alpha-agglutinin were bound to the C-terminal of GFPuv for display on the cell surface. In this novel system, the insertion of the recognition sequence-encoding gene of protease factor Xa between GFPuv and alpha-agglutinin was successfully carried out. The GFPuv, displayed by the insertion, was successfully cleaved from yeast cell surface by treatment with factor Xa, and could be easily recovered. By removing such a protease with well-known properties, the displayed protein could be isolated and purified with relative ease.

Magnetic properties of Heusler alloys Ru2-xFexCrSi.

We report on the structural and magnetic properties of newly synthesized Heusler alloys, Ru2-xFexCrSi, which have quite recently been shown to be candidates for ferromagnetic metals with high spin polarization from band structure calculations. Polycrystalline samples of Heusler alloys Ru2-xFexCrSi were prepared for 0.5≤x≤1.8. They were found to have L21 structures for 0.5≤x≤1.5 and B2 for x = 1.8. Magnetic measurements showed that they are ferromagnets. The Curie temperature for x = 1.0 was found to be 370 K. The Curie temperature tends to increase with increasing Fe concentration x. The saturation magnetic moment increases almost linearly as x increases. For higher Fe concentration the saturation magnetic moment is close to 2 µB per formula unit, which is theoretically expected.

Relationship between redox behavior of brain cytochrome oxidase and neurological prognosis.

Currently, no on-line method of assessing cerebral oxygenation is sufficiently accurate to be clinically helpful. In an attempt to find a good predictor of postoperative cerebral outcome, we retrospectively studied the relationship between the redox behavior of cytochrome oxidase (cyt. ox.) during an operation and the neurological prognosis in 83 patients who underwent thoracic aortic surgery. Our data revealed three patterns of change in the redox behavior of cyt. ox. during the operation; the actual pattern exhibited by a given patient showed a highly significant correlation with the neurological prognosis (p < 0.0001). We conclude that the redox behavior of cyt. ox. during an operation is likely to be a good predictor of postoperative cerebral outcome, which implies that brain tissue oxygen sufficiency can be evaluated by near-infrared measurement of cytochrome oxidase (except for that in local regions far from the monitoring site).

An epidemiologic survey of methicillin-resistant Staphylococcus aureus by combined use of mec-HVR genotyping and toxin genotyping in a university hospital in Japan.

OBJECTIVE: To evaluate the usefulness of an assay using two polymerase chain reaction-based genotyping methods in the practical surveillance of methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Nosocomial infection and colonization were surveyed monthly in a university hospital in Japan for 20 months. Genotyping with mec-HVR is based on the size of the mec-associated hypervariable region amplified by polymerase chain reaction. Toxin genotyping uses a multiplex polymerase chain reaction method to amplify eight staphylococcal toxin genes. RESULTS: Eight hundred nine MRSA isolates were classified into 49 genotypes. We observed differing prevalences of genotypes for different hospital wards, and could rapidly demonstrate the similarity of genotype for outbreak isolates. The incidence of genotype D: SEC/TSST1 was significantly higher in isolates causing nosocomial infections (49.5%; 48 of 97) than in nasal isolates (31.4%; 54 of 172) (P = .004), suggesting that this genotype may represent the nosocomial strains. CONCLUSION: The combined use of these two genotyping methods resulted in improved discriminatory ability and should be further investigated.

Redox behavior of cytochrome oxidase and neurological prognosis in 66 patients who underwent thoracic aortic surgery.

OBJECTIVE: Using near-infrared spectroscopy (NIRS), we have developed a new approach to the measurement of the redox state of cytochrome oxidase (cyt. ox.) in the brain. Our previous animal study showed that oxygen-dependent redox changes in cyt. ox. occur only when oxygen delivery is badly impaired. Therefore, in this study, we retrospectively examined the relationship between the redox behavior of cyt. ox. (measured by NIRS) during an operation and the neurological outcome in patients. METHODS: We studied 66 patients undergoing thoracic aortic surgery with cardiopulmonary bypass. Cerebral oxygenation was monitored by NIRS, and relative values for the concentrations of oxy-Hb, deoxy-Hb, and the redox state of cyt. ox. in the brain were calculated using our developed algorithm. RESULTS: Retrospective assessment revealed three different types of cyt. ox. behavior: (1) no change (type-A) in 34 cases (51.5%), (2) a temporary reduction, with a subsequent return to the pre-surgery baseline level (type-B) in 29 cases (43.9%), or (3) a marked and prolonged reduction (type-C) in only three cases (4.5%). Nine of the 66 patients (13.6%; one type-A, five type-B, and all three type-C patients) showed evidence of postoperative brain injury (in the type-A patient, the injury proved to be localized and far from the monitoring site). The relationship between the occurrence of such an injury and the type of cyt. ox. behavior seen during the operation was highly significant (P<0.0001; chi-square test for independence). CONCLUSIONS: Our data suggest that the redox behavior of cyt. ox. during an operation is a good (though not perfect) predictor of postoperative cerebral outcome, and that overall tissue oxygen sufficiency can be confirmed by near-infrared measurement of cyt. ox.