Improvements in the cell-free production of functional antibodies using cell extract from protease-deficient Escherichia coli mutant

Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expression system has been reported previously [Jiang et al., FEBS Lett., 514, 290-294 (2002)]. The low yield of the synthesized antibody, however, limits the usefulness of the cell-free expression system, partly due to the degradation of product by endogenous proteases from the E. coli extract. To determine which proteases are responsible for the degradation, we compared the expression of a 6D9 Fab fragment under conditions whereby several protease inhibitors were added into the cell-free system. The addition of serine protease inhibitor increased the amount of the Fab fragment, indicating that serine proteases caused the antibody degradation. Therefore, several serine protease-deficient mutants of E. coli BW25113 were constructed by targeted homologous recombination. The use of extract from a double protease-deficient mutant (DeltadegP-ompT) significantly increased the amount and antigen-binding activities of an anti-HSA scFv and a 6D9 Fab fragment. These results suggest that the DegP- and OmpT-deleted mutant is a useful source of S30 extract for the production or screening of antibodies using the cell-free expression system.     

Site-directed mutagenesis study of the antibody 2D7 which catalyzes a reaction for insertion of Cu2+ into mesoporphyrin

Monoclonal antibody 2D7 generated against a transition-state analog N-methyl mesoporphyrin catalyzes a reaction for insertion of a cupric ion into mesoporphyrin. To investigate amino acid residues responsible for the catalytic activity, site-directed mutagenesis of the amino acid residues in the third complementarity determining region of the heavy chain (CDRH3) was performed on the antigen-binding fragment (Fab) of the antibody. Recombinant Fab mutants, in which Arg95 is replaced with Ala (R95A), Asp96 with Asn (D96N) and Met97 with Gly (M97G), were examined in terms of the catalytic efficiency of the reaction (k/K(S)) and the dissociation constant for N-methyl mesoporphyrin binding (K(d)) and these values were compared with those of the wild type. The k/K(S) values of the R95A and D96N mutants were 0.96% and 1.0% of that of the wild type, respectively, whereas the M97G mutant had no detectable catalytic activity. The K(d) values of the R95A and D96N mutants were 165 and 69 times that of the wild type, respectively, while that of the M97G mutant was similar to that of the wild type. The relationship between the k/K(S) and 1/K(d) values in the wild type and the R95A and D96N mutants suggests that Arg95 and Asp96 are responsible for stabilizing the transition-state in the catalytic reaction. The results of the M97G mutant allow us to propose that Met97 plays an important role in the catalytic activity probably due to a subtle and specific conformation of the antibody.     

Shear cell development for diffusion experiments in FOTON-Satellite missions and on the ground with consideration of shear-induced convection

A shear cell technique was developed to obtain exact diffusion data. The shear cell in this study was designed for the utilization under μg-conditions, especially in the FOTON-M2 mission, but also under 1g-conditions. To minimize the influence of the shear convection, the cell size, the rotation system and the speed of the discs were optimized. To minimize free surfaces, which can cause Marangoni convection, a reservoir system providing pressure on the liquid was introduced. Using this FOTON shear cell we performed short-time diffusion experiments in the In-Sn system in a parabolic flight and under 1g conditions to investigate the influence of the shear convection quantitatively. As a result, the influence of the shear convection was so small that the mean square diffusion depth caused by the shear convection was in the order of10^{? 7}m², which is smaller than 1% of the typical value X _diff ² ≈ 10^{? 4}m² in a standard diffusion experiment using the FOTON shear cell. By using this result a correction method for the evaluation of the diffusion coefficient was established. In several ground experiments, the FOTON shear cell showed the same diffusion coefficients as from μg reference experiments within the range of errors and no obvious indication of Marangoni convection was detected. From these results we confirmed that the FOTON shear cell can be applied to μg-experiments and ground-based experiments as well.     

Self-organized inorganic nanoparticle arrays on protein lattices

Cavities formed by proteins have been utilized as the reaction chamber for the fabrication of a range of inorganic nanoparticles, providing control of the size of particles by limiting growth and preventing agglomeration. In crystal form, proteins construct molecular arrays that can provide regularly arranged sites for nanoparticles. Here we report the fabrication of nanometric iron and indium particles using ferritin, an iron-storage protein. The indium nanoparticles thus formed have uniform spherical shape with diameter of 6.6 +/- 0.5 nm, while the iron nanoparticles are somewhat irregular in shape (5.8 +/- 1.0 nm). Regular two-dimensional arrays of these nanoparticles are successfully produced by crystallizing ferritin molecules on a water-air interface using the denatured protein film method. The lattice constant of these nanoparticle arrays is 13 nm with hexagonal packing, and arrays of more than 1 microm in area can be obtained by transfer onto silicon wafer.     

Observation of HOPG by STM and contact AFM in various gas atmospheres under pressures up to 1.1 MPa

Apparatus comprising a scanning tunneling microscopy (STM) and an atomic force microscopy (AFM) has been developed for use under supra-atmospheres. Observations of highly oriented pyrolytic graphite (HOPG) were carried out by STM and contact AFM operating in air and various gas atmospheres (hydrogen, helium, neon and argon) under pressures up to 1.1 MPa. Atomic resolution images of the HOPG were obtained by STM in all the gas atmospheres studied. However, it was found that the presence of water vapor gave rise to a noise current at increased pressures. Using contact AFM, the atomic resolution in an argon atmosphere decreased with increasing pressure, while atomic images were obtained under the other gas atmospheres at 1.1 MPa.     

Behaviour of HCH (1,2,3,4,5,6-hexachlorocyclohexane) residue in the aquatic environment

The monthly variation in HCH (1,2,3,4,5,6-hexachlorocyclohexane) residue levels in surface waters was investigated. The logarithms of monthly HCH concentrations described the wave, and the equation of the wave was calculated by the least squares method. The waves indicated maximum concentrations in summer and minimum concentrations in winter at each sampling site. The maximum concentration of HCH in each year decreased gradually. The amplitude of the wave at the reservoir was the smallest among sampling sites. The slope of the wave at the downstream sampling site was smaller than that at the upper stream. Maximum concentration of total HCH at each sampling site in 1985 is estimated from the equations; the concentrations at the upper streams will be about a thousandth or a hundredth of that at the downstreams. Also, the equations for β-and γ-HCH were calculated, and indicated that β-HCH was more persistent than γ-HCH in surface water. The ratio of β-HCH to total HCH in surface water is estimated to be about 60% in 1985.     

Structural,electrical and optical properties of r.f.-magnetron-sputtered SnO2:Sb film

The effects of gas composition, pressure and substrate temperature on the properties of relatively thick (0.2–0.8 μm) SnO₂ films deposited onto fused quartz substrates by r.f. magnetron sputtering are reported. The lowest resistivity of about 2 × 10^?3ωcm was attained for high rate deposition conditions of about $\text{1000}\text{A}\text{?}\text{min}{}^{\mathrm{?1}}$ on substrates at a temperature of 400°C in an atmosphere of 10% O₂. This value corresponds to a carrier density of 3 × 10²⁰cm^?3 and a mobility of 10 cm²V^?1s^?1. The crystal structure was found to be sensitive to all the above parameters. Low resistivity films showed a highly preferred orientation of (101) parallel to the substrate.