METHOD OF IDENTIFYING REGULATOR SHORT-LIVED PROTEINS IN HUMAN PROTEOME INVOLVED IN SPECIFIC MODULATION OF CYTOTOXIC EFFECT Russian patent published in 2016 - IPC C12N15/62 C07K19/00 

FIELD: biochemistry.

SUBSTANCE: chimeric fusion proteins are obtained: GST USD HDAC6 and GST-PSMA3, via expression thereof in E. coli cells by adding corresponding vectors and subsequent treatment by affine chromatography on glutathione-sepharose. Method comprises reaction of binding proteins from extract of multiple myeloma cells before or after combined chemotherapy with bortezomib and doxorubicin with GST-UBD_HDAC6 and GST-PSMA3. Method includes washing off bound proteins from chromatographic carrier with glutathione-containing buffer solution. Desalination and concentration are performed on micro-concentrators. Obtained protein preparations are separated in a two-dimensional electrophoresis system: I direction - isoelectric focusing of proteins on device Ettan IPG Phor3 (GE Healthcare), II direction - separation of proteins by molecular mass in denaturating Laemmli electrophoresis. Spots corresponding to separate proteins are cut from polyacrylamide gel and composition thereof is analysed. Higher efficiency of cleaning regulatory of short-lived proteins (both polyubiquitinated (GST-UBD_HDAC6) and subject to ubiquitin-independent proteolysis GST-PSMA3)). Convenience of method consists in that it makes it possible to manipulate directly with cell extracts, without additional purification steps of proteins.

EFFECT: invention simplifies diagnosis of multiple myeloma by detecting among short-lived regulatory proteins specific markers specific for given disease.

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