Expression of SSX2 tumor antigen in baculovirus expression system and its application for screening of blood serum of melanoma patients.

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.

Molecular chaperone, HSP60, and cytochrome P450 2E1 co-expression in dilated cardiomyopathy.

Physiological stresses (heat, hemodynamics, genetic mutations, oxidative injury and myocardial ischemia) produce pathological states in which protein damage and misfolded protein structures are a common denominator. The specialized proteins family of antistress proteins - molecular chaperons (HSPs) - are responsible for correct protein folding, dissociating protein aggregates and transport of newly synthesized polypeptides to the target organelles for final packaging, degradation or repair. They are inducible at different cell processes such as cell division, apoptosis, signal transduction, cell differentiation and hormonal stimulation.

Immunohistochemical analysis of S6K1 and S6K2 expression in human breast tumors.

Aim: To express recombinant S6K2 in baculovirus expression system; to purify large quantities of recombinant S6K2 for biochemical studies; to generate and characterise specific MABs against recombinant S6K2; to study the patterns S6K1 and S6K2 expression and subcellular localization in normal, benign and malignant breast tissues.

Methods: Recombinant baculovirus, expressing wild type S6K2 was generated using Bac-to-Bac system (Invitrogen); recombinant S6K was purified from infected Sf9 cells using affinity purification approach; monoclonal antibodies against recombinant S6K2 were generated; the specificity of generated MABs towards recombinant and endogenous S6K2 were examined by ELISA, Western blotting, immunoprecipitation and immuhohistochemical staining; immunohistochemical detection of S6K1 and S6K2 in human breast tissues was performed using specific monoclonal antibodies towards S6K1 and S6K2.

Results: Large amounts of enzymatically active S6K2 were purified using baculovirus expression system; highly purified preparations of S6K2 were used to generate and characterize anti-S6K2 MABs; elevated levels of S6K1 and S6K2 were found in breast tumors when compared to normal breast tissues; S6K2 is frequently localized in the nuclei of adenocarcinoma tissues, but rarely in fibroadenoma or "normal" breast tissues.

Subcellular localization and regulation of coenzyme A synthase.

CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme.