Generation of expression clone set for functional proteomics of human gastric and liver cancers.
Two thousand sixty-eight multi-purpose expression clones for the 326 candidate genes related to gastric or liver cancers were constructed using the Gateway system. These clones can be expressed as His, Glutathione-S-transferase (GST) or Enhanced version of the green fluorescent protein (EGFP) fusion proteins in E. coli, insect cells or mammalian cells. For the 246 E. coli expression clones, the GST fusion proteins had greater expression efficiency and solubility than the His fusion proteins. Approximately 20% of the expressed proteins had unexpected molecular weights. A detailed sequence analysis of these clones revealed frameshift mutations resulting from insertion, deletion or substitution of nucleotides. The results indicate that these changes in the candidate genes may affect the occurrence of gastric or liver cancers. In addition, when 105 proteins, which were expressed in E. coli at very low or undetectable levels, were expressed in insect cells, 76% of the proteins were expressed very well and most were soluble. We also found that most of the 30 proteins prepared using EGFP mammalian expression clones were localized to cellular compartments expected by Gene ontology (GO) and this localization was unaffected if the EGFP-fusion was at the N-terminal or C-terminal region of the protein. Antibody production and subcellular localization analysis of the candidate genes as well as a screen of genes involved in carcinogenesis pathways are currently in progress using these expression clones. These studies provide a valuable resource for developing a better understanding of the molecular mechanism of carcinogenesis in both gastric and liver cancer and would be very helpful in diagnosis and therapeutic predictions.
Oh
Nang-Soo
NS
Laboratory of Human Genomics, Genome Research Center, KRIBB, Daejeon 305-806, Korea.
Park
Ji-Seon
JS
Jeon
Yeo-Jin
YJ
Oh
Jung-Hwa
JH
Jeong
So-Young
SY
Yang
Jin-Ok
JO
Park
Yong-Won
YW
Yoo
Hyang-Sook
HS
Kim
Nam-Soon
NS
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D005456:Fluorescent Dyes; D011506:Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006639:Histidine; D005982:Glutathione Transferase