Membrane Protein Expression in Lactococcus lactis

Abstract

Membrane proteins play key roles in cellular physiology, and they are important drug targets. Approximately 25% of all genes identified in sequenced genomes are known to encode membrane proteins; however, the majority have no assigned function. Although the resolution of soluble protein structure has entered the high-throughput stage, only 100 high-resolution structures of membrane proteins have been described until now. Lactococcus lactis is a gram-positive lactic bacterium that has been used traditionally in food fermentations, but it is now used widely in biotechnology for large-scale overproduction of heterologously expressed proteins. Various expression vectors based on either constitutive or inducible promoters exist. The nisin-inducible controlled gene expression (NICE) system is the most suitable for recombinant membrane protein expression allowing for fine control of gene expression based on the autoregulation mechanism of the bacteriocin nisin. Recombinant membrane proteins can be produced with affinity tags for efficient detection and purification from crude membrane protein extracts. The purpose of this chapter is to provide a detailed protocol for the expression of membrane proteins and their detection using the Strep-tag II affinity tag in L. lactis.

Comparative genomics and functional analysis of the NiaP family uncover nicotinate transporters from bacteria, plants, and mammals

“…L. lactis is an efficient platform for functional expression of pro-and eukaryotic membrane proteins (Kunji et al 2003;Frelet-Barrand et al 2010) and a niacin auxotroph (Neves et al 2002). We first obtained direct evidence that B. subtilis NiaP transports nicotinate, and explored mechanism and substrate range.…”

Membrane Protein Expression in Lactococcus lactis

“…-Using the correct procedures (as detailed above), whole cells can be frozen and stored at À80°C without adverse effects. -The one-shot disruption system of Constant Systems is the most suitable system to disrupt the lactococcal cell wall as the yield of crude membranes improves more than fivefold when compared to that obtained by lysozyme and French press treatment (Frelet-Barrand, Boutigny, Kunji, et al, 2010).…”

“…As the natural levels of the majority of membrane proteins are low, overexpression is a prerequisite for functional and structural studies. In the last 10 years, the Gram-positive bacterium Lactococcus lactis has proven itself to be a good alternative for the overproduction of membrane proteins, often in a functional form (Frelet-Barrand, Boutigny, Kunji, & Rolland, 2010;Frelet-Barrand, Boutigny, Moyet, et al, 2010;Kunji et al, 2005;Kunji, Slotboom, & Poolman, 2003;. One of the major reasons is the development of the nisin-controlled expression system, which is based on a two-component regulatory system that is part of an operon involved in the production of the lantibiotic nisin A (de Ruyter, Kuipers, & de Vos, 1996).…”

Membrane Protein Production for Structural Analysis

“…The first one is a nonpathogenic and noninvasive lactic acid Gram-positive bacterium (for a review, see Junge et al 2008;Frelet-Barrand et al 2010a; see also Chap. 5 in this volume).…”