brij-58 and Leukemia--Lymphocytic--Chronic--B-Cell

The anti-apoptotic B cell CLL/lymphoma-2 (Bcl-2) protein is a key player in the regulation of programmed cell death and is linked to various types of cancer and their resistance to drug treatment. Biophysical and structural studies of the full-length intact Bcl-2 have been hampered due to difficulties in expression and severe solubility problems, precluding isolation of this hydrophobic membrane protein. Therefore, previous work has so far mainly been carried out using structurally modified Bcl-2 variants, lacking the transmembrane region. Thus, biophysical information regarding the full-length protein is still missing. Here, a protocol is presented for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)). A batch-based cell-free expression system, using extract isolated from Escherichia coli (E. coli) was employed to produce recombinant protein encoded by an optimized gene sequence. Presence of polyoxyethylene-(20)-cetyl-ether (Brij-58) in the reaction mixture and subsequently in the immobilized metal-affinity purification steps was crucial to keep Bcl-2(2) soluble. The obtained yield was 0.25-0.3mg per ml of cell-free reaction. Far-UV circular dichroism (CD) spectroscopy confirmed the α-helical structure of the purified protein, characteristic for members of the Bcl-2 protein family.

Topics: Apoptosis; Cell Fractionation; Cell-Free System; Cetomacrogol; Chromatography, Affinity; Circular Dichroism; Cloning, Molecular; Escherichia coli; Humans; Hydrophobic and Hydrophilic Interactions; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Protein Biosynthesis; Protein Folding; Protein Isoforms; Protein Structure, Secondary; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Solubility; Subcellular Fractions