Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

SummaryIn maturing seed cells, many newly synthesized proteins are transported to the protein storage vacuoles (PSVs) via vesicles unique to seed cells. Vacuolar sorting determinants (VSDs) in most of these proteins have been determined using leaf, root or suspension-cultured cells apart from seed cells. In this study, we examined the VSD of the a 0 subunit of b-conglycinin (7S globulin), one of the major seed storage proteins of soybean, using Arabidopsis and soybean seeds. The wild-type a 0 was transported to the matrix of the PSVs in seed cells of transgenic Arabidopsis, and it formed crystalloid-like structures. Some of the wild-type a 0 was also transported to the translucent compartments (TLCs) in the PSV presumed to be the globoid compartments. However, a derivative lacking the C-terminal 10 amino acids was not transported to the PSV matrix, and was secreted out of the cells, although a portion was also transported to the TLCs. The Cterminal region of a 0 was sufficient to transport a green fluorescent protein (GFP) to the PSV matrix. These indicate that a 0 contains two VSDs: one is present in the C-terminal 10 amino acids and is for the PSV matrix; and the other is for the TLC (the globoid compartment). We further verified that the C-terminal 10 amino acids were sufficient to transport GFP to the PSV matrix in soybean seed cells by using a transient expression system.

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Meiotic maturation is essential for the reproduction procedure of many animals. During this process an oocyte produces a large egg cell and tiny polar bodies by highly asymmetric division. In this study, to fully understand the sophisticated spatiotemporal regulation of accurate oocyte meiotic division, we focused on the global and local changes in the tension at the surface of the starfish (Asterina pectinifera) oocyte in relation to the surface actin remodeling. Before the onset of the bulge formation, the tension at the animal pole globally decreased, and started to increase after the onset of the bulge formation. Locally, at the onset of the bulge formation, tension at the top of the animal pole began to decrease, whereas that at the base of the bulge remarkably increased. As the bulge grew, the tension at the base of the bulge additionally increased. Such a change in the tension at the surface was similar to the changing pattern of actin distribution. Therefore, meiotic cell division was initiated by the bulging of the cortex, which had been weakened by actin reduction, and was followed by contraction at the base of the bulge, which had been reinforced by actin accumulation. The force generation system is assumed to allow the meiotic apparatus to move just under the membrane in the small polar body. Furthermore, a detailed comparison of the tension at the surface and the cortical actin distribution indicated another sophisticated feature, namely that the contraction at the base of the bulge was more vigorous than was presumed based on the actin distribution. These features of the force generation system will ensure the precise chromosome segregation necessary to produce a normal ovum with high accuracy in the meiotic maturation.