ABSTRACT
PTEN is a tumor suppressor with dual protein and lipid-phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202-NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K(D) of PTEN-FABP4 complex, which is around 2.8 microM.
Subject(s)
Fatty Acid-Binding Proteins/metabolism , PTEN Phosphohydrolase/metabolism , Proteins/isolation & purification , Proteins/metabolism , 3T3-L1 Cells , Animals , Carcinoma/metabolism , Colonic Neoplasms/metabolism , Embryo, Mammalian , Fatty Acid-Binding Proteins/isolation & purification , Gene Library , HeLa Cells , Humans , Mice , Protein Binding , Two-Hybrid System Techniques , YeastsABSTRACT
AIM: To clone, express in baculovirus expression system and purify SSX2 tumor antigen and to use it for screening of blood serum of melanoma patients. MATERIALS AND METHODS: Cloning and expression of SSX2 antigen in Bac-to-Bac baculovirus expression system as His-tag fusion protein, expression of recombinant SSX2 in insect cells with following purification by affinity chromatography on Ni-NTA agarose, ELISA of blood serum of melanoma patients (n = 29) and healthy donors (n = 27) were used. RESULTS: SSX2 was cloned in baculovirus expression system, expressed, purified using affinity chromatography and used in ELISA as antigen. Comparative analysis of blood serum of melanoma patients and healthy donors revealed higher level of SSX2-positivity of blood serum from the patients with cancer. CONCLUSION: SSX2 antigen expressed in baculovirus expression system can be used for serological analysis of blood serum of oncological patients.
Subject(s)
Antigens, Neoplasm/blood , Enzyme-Linked Immunosorbent Assay/methods , Melanoma/diagnosis , Neoplasm Proteins/blood , Repressor Proteins/blood , Skin Neoplasms/diagnosis , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/isolation & purification , Baculoviridae/genetics , Cloning, Molecular , Humans , Neoplasm Proteins/genetics , Neoplasm Proteins/isolation & purification , Repressor Proteins/genetics , Repressor Proteins/isolation & purificationABSTRACT
Genes rplJ, coding for ribosomal protein L10 of Salmonella typhimurium and Klebsiella pneumoniae, have been cloned on pUC plasmid. The resultant multicopy recombinant plasmids were detrimental for the growth of normal JM101 E. coli host cells and harmless for the mutant JF3029 host. This negative effect is the evidence for the ability of heterologous L10 proteins to regulate expression of rplJL genes in E. coli. Nucleotide sequence was determined completely for S. typhimurium rplJL' DNA portion and partially for rplJL' genes of K. pneumoniae. According to the nucleotide sequence data obtained three amino acid substitutions differ L10 proteins of S. typhimurium and E. coli and the long range, providing for the coupled translations of L10 and L7/L12 cistrons in E. coli mRNA is also valid for S. typhimurium and K. pneumoniae.
