A high-level secretion system for the production of mouse-human chimeric antibody 21B2 (MHC 21B2) Fab fragment specific for human carcino embryonic antigen (hCEA) in Escherichia coli has been constructed. The genes encoding a light chain and an Fd fragment (a variable region and the CH1 domain of a heavy chain) of a mouse-human chimeric antibody were directly fused to the signal peptide of the E. coli ompF gene sequence. E. coli cells containing expression vectors in which each of the two genes are located downstream of a separate tac promoter were able to secrete the light chain and Fd fragment as two of their major cellular proteins. The signal peptides were efficiently removed from the primary products by post-translational processing, although they formed insoluble aggregates, possibly in the periplasm. In high-cell-density culture experiments using a jar fermentor, the amount of light chain and Fd fragment produced was at levels of up to 2.88 g/l and 1.28 g/l culture, respectively. By optimizing the conditions that encourage correct folding, formation of disulphide bonds, and association of the light chain with the Fd fragment, we have established a procedure that can purify, re-fold, and combine aggregated products to electrophoretically homogeneous Fab fragment with a yield of approximately 47%. Fab fragment produced in this manner shows essentially the same antigen-binding activity and specificity to hCEA as the parental mouse antibody 21B2 (MoAb 21B2).
The conformation change of Thermus thermophilus tRNA~ le upon complex formation with T. thermophilus elongation factor Tu (EF-Tu) was studied by analysis of the circular dichroism (CD) bands at 315 nm (due to the 2-thioribothymidine residue in the T-loop) and at 295 nm (due to the core structure of tRNA). Formation of -Ile the ternary complex of lsoleucyl-tRNA 1 and EF-Tu.GTP increased the intensities of these CD bands, indicating stabilization of the association between the T-loop and the D-loop and also a significant conformation change of the core region. Upon complex formation of EF-Tu.GTP and uncharged tRNA, however, the conformation of the core region is not changed, while the association of the two loops is still stabilized. On the other hand, the binding with EF-Tu.GDP does not appreciably affect the conformation of isoleucyl-tRNA or uncharged tRNA. These indicate the importance of the y-phosphate group of GTP and the aminoacyl group in the formation of the active complex of aminoacyl-tRNA and EF-Tu-GTP.
In this study, Nb doped γ-TiAl is designed and examined their tensile properties at elevated temperatures. Small compositional changes cause drastic changes of the mechanical properties at 1273K. In order to clarify the deformation mechanism at elevated temperatures, dislocation structures have been observed using transmission electron microscope (TEM). All of the specimens observed by TEM show that at least two slip systems are operating in each grain. The specimen having relatively lower strength and higher tensile elongations shows more than four operating slip systems in the grains. The specimen having a medium strength shows many super-dislocations and their dissociations that block the other dislocation motions. The specimen having the highest strength shows many deformation twins. These observations suggest that deformation twins become the strong obstacles against moving dislocations at elevated temperatures. The relationships between observed dislocation structures and mechanical properties of γ-TiAl are discussed.
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