Beta-galactosidase fused to the hydrophobic domain of cytochrome b5 spontaneously associates with liposomes.
Since liver microsomal cytochrome b5 spontaneously associates with liposomes and membranes by means of its C-terminal hydrophobic domain (HP), chimeric proteins containing HP prepared by genetic fusion might also spontaneously associate with liposomes or cellular membranes. Synthetic DNA corresponding to the hydrophobic domain of cytochrome b5 was enzymatically fused in-frame to cloned DNA corresponding to the C-terminus of the Escherichia coli enzyme, beta-galactosidase. This protein, LacZ:HP, synthesized in E. coli and purified from a crude E. coli membrane extract, was shown to spontaneously associated with liposomes, as does cytochrome b5. Association is rapid and stable in the presence of salt and high pH and the fusion protein behaves as an integral membrane protein. LacZ:HP can be readily and extensively purified from crude extracts by association with liposomes and this procedure may provide a convenient purification scheme for proteins not otherwise readily purified, for example polypeptides from cloned gene fragments to be used for antibody production. These hybrid proteins may represent a new potentially useful class of polypeptides capable of hydrophobic interactions with membranes.
George
S K
SK
Department of Medical Biochemistry and Genetics, Texas A & M College of Medicine, College Station 77843.
Najera
L
L
Sandoval
R
R
Ihler
G M
GM
D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D015786:Cytochromes b5 / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D008081:Liposomes; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5; D001616:beta-Galactosidase