Cell-free translation of integral membrane proteins into unilamelar liposomes.
Wheat germ cell-free translation is shown to be an effective method to produce integral membrane proteins in the presence of unilamelar liposomes. In this chapter, we describe the expression vectors, preparation of mRNA, two types of cell-free translation reactions performed in the presence of liposomes, a simple and highly efficient purification of intact proteoliposomes using density gradient ultracentrifugation, and some of the types of characterization studies that are facilitated by this facile preparative approach. The in vitro transfer of newly translated, membrane proteins into liposomes compatible with direct measurements of their catalytic function is contrasted with existing approaches to extract membrane proteins from biological membranes using detergents and subsequently transfer them back to liposomes for functional studies.
Goren
Michael A
MA
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Nozawa
Akira
A
Makino
Shin-ichi
S
Wrobel
Russell L
RL
Fox
Brian G
BG
D000818:Animals; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008954:Models, Biological; D014176:Protein Biosynthesis / Q000502:physiology; D014170:Transformation, Genetic / Q000502:physiology; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D008565:Membrane Proteins; D053835:Unilamellar Liposomes