Back to Search
Start Over
Generation of Expression Clone Set for Functional Proteomics of Human Gastric and Liver Cancers
- Source :
- Experimental Biology and Medicine. 234:1220-1229
- Publication Year :
- 2009
- Publisher :
- SAGE Publications, 2009.
-
Abstract
- 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.
- Subjects :
- Proteomics
Candidate gene
Sequence analysis
Recombinant Fusion Proteins
Green Fluorescent Proteins
Spodoptera
Biology
Kidney
Transfection
medicine.disease_cause
General Biochemistry, Genetics and Molecular Biology
Cell Line
Green fluorescent protein
Frameshift mutation
Mice
Stomach Neoplasms
Escherichia coli
medicine
Animals
Humans
Histidine
Cloning, Molecular
Gene
Cells, Cultured
Fluorescent Dyes
Glutathione Transferase
Liver Neoplasms
Proteins
Subcellular localization
Molecular biology
Fusion protein
Solubility
NIH 3T3 Cells
Carcinogenesis
Subcellular Fractions
Subjects
Details
- ISSN :
- 15353699 and 15353702
- Volume :
- 234
- Database :
- OpenAIRE
- Journal :
- Experimental Biology and Medicine
- Accession number :
- edsair.doi.dedup.....9d328ba647bc8e16d1216e6ebd240d2d
- Full Text :
- https://doi.org/10.3181/0812-rm-371