The Ultrastructure Of Dictyostelium Discoideum AK127 Cells And Expression, Protein Purification Of Gp150

Dictyostelium discoideum is a kind of lower unicellular eukaryote,which multiplies by binary fission under favorable conditions. Once starving or depleting their food source,multicellular development will be induced, the cell will undergo a patyway including aggregation, mound, slug, culmination stages, forming a fruiting body consisted of a head of spores and a stalk finally,the procedure of cell developm-ent is regulated exactly. Dictyostelium discoideum is regarded as an excellent system to study cell-cell signal transduction,cell development,cell type differentiation for its short life cycle and rich life phenomenon.The transmembrane glycoprotein gp150 expressed in mound stage is absolutely necessary in cell development, this adhesion molecule gp150 play a role in cell-type specification by heterophilic interaction. A mutant strain in which lagC is knocked out,AK127,undergoes normal chemotaxis, but is arrested at the loose aggregate stage. We inferred that gp150 is a key factor in the whole cell development,however,the concrete mechanism of sigal transduction which gp150 participated in.In order to understand the effect of adhesion molecule gp150, AK127 cells after 14 h,16 h and 20 h of development were studied by transmission electron microscopy respectively,.The result shows that after 14 h of development,The density of cytoplasm enclosed by some rough endoplasmic reticulum is more transparent than cytoplasm around. Abundant multi-membrane structure can be observed clearly. We inferred that these multi-membrane structures may be originated from rough endoplasmic reticulum. After 16 h of development, some membrane of multi-membrane structure commence to disrupt, several autophagic vacuoles have emerged from these multi-membrane structures..After 20 h of development,a large autophagic vacuole containing several multi-membrane structures can be observed. The author presumes that these multi-membrane structures may be regarded as "warehouses" where nutrient been reserved, so that cell life can be maintained. Because of inner nuclear membrane depression, some hollow spaces containing filamentous material exist between out nuclear membrane and inner nuclear membrane of nucleus. Some spiral chromatin is suspended from inner nuclear membrane and out nuclear membrane is covered by granular materials.Some round vacuole covered by granular material occoured around nucleus mentioned above, However, this phenomenon had not be seen around nucleus in which karyotheca remained intact.The author presumed that these round vacuoles probably originated from hollowed nucleus. Cristae in mitochondria were intact at 14 h,16 h and 20 h of development,rather than endoautophagy occuring at organelle level in wild strain. The results suggest that ultrastructural characters of cells may have some relationship with their special physiological status.Besides the ultrastructural observation of AK127, genetic engineering was adopted for studying the structure and function of gp150.To obtain enough purified protein for crystal structure research, prokaryotic expression method was used.We transformed the vector Actin15-LagC into DH5a and expressed in E. coli BL21 (DE3) host cells. After the expression strain was induced for 4 hours by 1mmol/L IPTG, gp150 was successly expressed and detected in soluble fraction.The protein band in SDS-PAGE showed that gp150 accounted 15% of total bacterial protein. Three kind of ion exchange chromatography(CM,DEAE,Q)were applied in protein purification of gp150 under different pH value.Due to vector Actin15-LagC was lack of fusion tags,the effect of protein purification failed to reach the standard as expected.Then we cloned the LagC by PCR from fomer vector Actin15-LagC which as template.The LagC gene of Dictyostelium discoideum was susscessfully cloned into the fusion expression vector pET-32a which contain His-Tag. We got recombinant vector which supplies good tool for researching the structure of gp150 and for studying its function during dictyostelium development.The secondary structure of gp150 were predicted by the methods of Chou-Fasman,Gamier-Robson.Moreover,flexible regions,hydrophilicity plot, surface probability plot and antigenic index of gp150 were predicted by the methods of Karplus-Schulz,Kyte-Doolittle,Emini and Jameson-Wolf,respectively.According to the results,we can conclude that gp150 protein contained moreβsheet regions. There were several centers of a helix in the regions of 348-353,598～602,685～688, 731～732,733～734,and several centers ofβsheet in more than 40 regions:5-16, 26～30,34～40,46～52,69～72,98～101,116～123 etc. Moreover,many distinct epitopes in gp150 possibly localized in the regions of 19～20,31～34,,72～76,101～105, 264～267,371373,517～521,697～699.These results will be helpful for estimating of the functional domains and provide a theory basis for further research of gp150.