[Expression, purification and activity analyses of three Bcl-2 family proteins].
Bcel-2 family proteins (Bcl-x(L), Bcl-2, Mel-1 etc.) are key regulators of some life processes, including apoptosis and autophagy. They are currently considered as promising targets for developing new anti-tumor therapies. In our study, the human Bcl-2/Bcl-x(L) chimeric gene and the human/mouse Mel-1 chimeric gene were designed and cloned, and the prokaryotic expression vectors for expressing glutathione S-transferase (GST) fusion proteins and histidine tag fusion proteins were constructed respectively. These two proteins as well as the GST-Bcl-x(L) fusion protein were all successfully expressed in E. coli and subsequently purified. In addition, we measured the binding of these Bcl-2 family proteins to the Bid BH3 peptide by fluorescence polarization-based assay. The dissociation constants (Kd) obtained by us were in general agreement with the data reported in literature. The Kd values of all three proteins with or without the GST tag were almost identical. All these results validate the biological functions of these Bcl-2 family proteins obtained by us. These proteins can be used in the experimental screening of small-molecule regulators of Bcl-2 family proteins in vitro.
Zhu
Cuixia
C
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China.
Li
Xun
X
Li
Wenwen
W
Shi
Zhimin
Z
Zhou
Jiahai
J
Wang
Renxiao
R
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D051020:bcl-X Protein / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein; D005982:Glutathione Transferase