Functional in vitro assembly of the integral membrane bacterial thermosensor DesK

MARIANA MARTÍN A, DANIELA ALBANESI A,B, PEDRO M. ALZARI B, DIEGO DE MENDOZA A,*

PROTEIN EXPRESSION AND PURIFICATION

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 2009 vol. 66 p. 39 - 45

1046-5928

The Bacillus subtilis DesK histidine kinase (HK) is an integral membrane thermosensor that forms part of a regulatory circuit which controls the physical state of membrane lipids. In the pursuit of biochemical and structural approaches to study lipid fluidity-dependent DesK thermosensing, we found that standard expression methods failed to produce enough amounts of a fully functional protein. Here, we describe a high-yield purification method based in an Escherichia coli in vitro transcription–translation system. The enzymatic activities of the full-length protein, either solubilized with detergents or co-translationally inserted into liposomes, have been characterized and compared with those measured for the constitutively active cytoplasmic domain of DesK, lacking the transmembrane sensor domain. As expected, the autokinase activity of liposome-inserted DesK was greatly increased when the incubation temperature was decreased from 37 to 25 C. This is the first report of the spontaneous in vitro membrane insertion of a fully functional bacterial HK thermosensor. Moreover, this single step procedure should greatly aid the isolation of a wide range of membrane-associated HKs for biochemical and biophysical studies.Bacillus subtilis DesK histidine kinase (HK) is an integral membrane thermosensor that forms part of a regulatory circuit which controls the physical state of membrane lipids. In the pursuit of biochemical and structural approaches to study lipid fluidity-dependent DesK thermosensing, we found that standard expression methods failed to produce enough amounts of a fully functional protein. Here, we describe a high-yield purification method based in an Escherichia coli in vitro transcription–translation system. The enzymatic activities of the full-length protein, either solubilized with detergents or co-translationally inserted into liposomes, have been characterized and compared with those measured for the constitutively active cytoplasmic domain of DesK, lacking the transmembrane sensor domain. As expected, the autokinase activity of liposome-inserted DesK was greatly increased when the incubation temperature was decreased from 37 to 25 C. This is the first report of the spontaneous in vitro membrane insertion of a fully functional bacterial HK thermosensor. Moreover, this single step procedure should greatly aid the isolation of a wide range of membrane-associated HKs for biochemical and biophysical studies.Escherichia coli in vitro transcription–translation system. The enzymatic activities of the full-length protein, either solubilized with detergents or co-translationally inserted into liposomes, have been characterized and compared with those measured for the constitutively active cytoplasmic domain of DesK, lacking the transmembrane sensor domain. As expected, the autokinase activity of liposome-inserted DesK was greatly increased when the incubation temperature was decreased from 37 to 25 C. This is the first report of the spontaneous in vitro membrane insertion of a fully functional bacterial HK thermosensor. Moreover, this single step procedure should greatly aid the isolation of a wide range of membrane-associated HKs for biochemical and biophysical studies.in vitro membrane insertion of a fully functional bacterial HK thermosensor. Moreover, this single step procedure should greatly aid the isolation of a wide range of membrane-associated HKs for biochemical and biophysical studies.