Lactococcus lactis as host for overproduction of functional membrane proteins.
Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. The organism is easy and inexpensive to culture, has a single membrane and relatively mild proteolytic activity. Methods for genetic manipulation are fully established and a tightly controlled promoter system is available, with which the level of expression can be varied with the inducer concentration. Here we describe our experiences with lactococcal expression of the mechanosensitive channel, the human KDEL receptor and transporters belonging to the ABC transporter family, the major facilitator superfamily, the mitochondrial carrier family and the peptide transporter family. Previously published expression studies only deal with the overexpression of prokaryotic membrane proteins, but in this paper, experimental data are presented for the overproduction of mitochondrial and hydrogenosomal carriers and the human KDEL receptor. These eukaryotic membrane proteins were expressed in a functional form and at levels amenable to structural work.
Kunji
Edmund R S
ER
MRC Dunn Human Nutrition Unit, Hills Road, CB2 2XY Cambridge, UK. ek@mrc-dunn.cam.ac.uk
Slotboom
Dirk-Jan
DJ
Poolman
Bert
B
D000818:Animals; D005057:Eukaryotic Cells; D015870:Gene Expression; D006801:Humans; D013294:Lactococcus lactis / Q000254:growth & development / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000226:Mitochondrial ADP, ATP Translocases / Q000096:biosynthesis; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009561:Nisin / Q000096:biosynthesis; D010957:Plasmids; D011485:Protein Binding; D018000:Receptors, Peptide / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis
C064459:KDEL receptor; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; C112225:PET9 protein, S cerevisiae; D018000:Receptors, Peptide; D029701:Saccharomyces cerevisiae Proteins; D009561:Nisin; D000226:Mitochondrial ADP, ATP Translocases; C102526:nisin A