Lactococcus lactis as host for overproduction of functional membrane proteins

Abstract

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.

Agmatine deiminase pathway genes in Lactobacillus brevis are linked to the tyrosine decarboxylation operon in a putative acid resistance locus

“…The AgmP PCR product was digested with NcoI and XbaI and inserted into the corresponding sites of the Lc. lactis expression vector pNZ8048 (Kunji et al, 2003). Sequencing confirmed the presence of the aguA1, aguA2 and aguD genes downstream of an in-frame sequence encoding an N-terminal histidine tag in plasmids pET/ aguA1, pET/aguA2 and pNZAgmP, respectively.…”

Heterologous expression of pneumococcal virulence factor PspC on the surface of Lactococcus lactis confers adhesive properties

“…The non-pathogenic Lactococcus lactis has been shown to be an optimal organism to express and decipher the function of prokaryotic and eukaryotic proteins (Kunji et al, 2003(Kunji et al, , 2005Monné et al, 2005;Morello et al, 2008 …”

“…The non-pathogenic Lactococcus lactis has been shown to be an optimal organism to express and decipher the function of prokaryotic and eukaryotic proteins (Kunji et al, 2003(Kunji et al, , 2005Monné et al, 2005;Morello et al, 2008). L. lactis was further reported to be more advantageous an expression host than Escherichia coli (Mierau & Kleerebezem, 2005;Zhou et al, 2006) and, with regard to pneumococcal PspC, the use of lactococci is preferred as PspC is again decorated on a Gram-positive cell surface.…”

“…Hence, the effects or influences of other pneumococcal proteins on, for example, adherence or induced host immune responses cannot be fully excluded. Therefore, we intended to study the effects of PspC under conditions in which none of the other pneumococcal virulence factors or adhesins may influence the course of infection.The non-pathogenic Lactococcus lactis has been shown to be an optimal organism to express and decipher the function of prokaryotic and eukaryotic proteins (Kunji et al, 2003(Kunji et al, , 2005Monné et al, 2005;Morello et al, 2008 …”

Mapping the yeast host cell response to recombinant membrane protein production: Relieving the biological bottlenecks

“…The authors further speculated that low yields of recombinant membrane proteins might be due to limited Sec translocon capacity. A second prokaryote, the Gram-positive bacterium Lactococcus lactis, has also been used to produce a wide range of eukaryotic and prokaryotic membrane proteins [16], enabling a comparison of L. lactis and E. coli [17]. Although a large fraction of proteins could be produced in both hosts, some could only be produced in one or the other.…”