Elimination of false negative results in the two-hybrid system in the phagocyte NADPH oxidase.
The yeast two-hybrid system is finding increased use in the study of interactions between proteins. In this method, two polypeptides are expressed in yeast as fusion proteins to a transcriptional activator DNA-binding domain (bd) and activating domain (ad), respectively. Interaction between the two polypeptides reconstitutes function of a transactivator which controls expression of reporters. The phagocyte NADPH oxidase is a complex of membrane cytochrome b558 (comprised of subunits p22-phox and gp91-phox) and three cytosol proteins (p47-phox, p67-phox, and p21rac) that translocate to membrane and bind to cytochrome b558. This is the first report to demonstrate that two of cytosolic components of cytochrome b558, p47-phox binding to p67-phox each other. We encountered several methodological problems in the two-hybrid system which are the focus of this report.
Hong
Y R
YR
Graduate Institute of Biochemistry, Kaohsiung Medical College, Taiwan, Republic of China.
Han
L
L
Huang
J
J
Kleinberg
M E
ME
D005188:False Negative Reactions; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis*; D019255:NADPH Oxidases; D010586:Phagocytes / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases