Lactococcus lactis, an Alternative System for Functional Expression of Peripheral and Intrinsic Arabidopsis Membrane Proteins

Abstract

BackgroundDespite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.Methodology/Principal FindingsThe aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.Conclusions/SignificanceAltogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.

Biochemical Characterization of AtHMA6/PAA1, a Chloroplast Envelope Cu(I)-ATPase

“…In the transformed L. lactis, PAA1 was found to stand for 2-3% of the total membrane proteins (26). As shown in Fig.…”

“…In this mutant, the aspartate residue of the DKTGT consensus sequence was replaced by an alanine, a substitution known to prevent the transient phosphorylation of any P-type ATPase. Both PAA1 and PAA1-AKT contain a Strep-Tag II (IBA, Goettingen, Germany) at their C-terminal end (26).…”

“…This is mainly explained by the difficulty to get high amounts of these membrane proteins in an active form either from their native environment or after expression in heterologous systems. Recently, we have shown that Lactococcus lactis, a Gram-positive lactic bacterium, is an attractive system for efficient production of plant membrane proteins and especially for P IB -type ATPases; AtHMA1, AtHMA3, and PAA1, produced at 300 -900 g/liter, account for 1-3% of the total membrane proteins (26). Thus, this heterologous system provides the unique opportunity to get access to enzymatic parameters of these plant ATPases, parameters that cannot be apprehended by the characterization of plant mutants alone.…”

BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

“…Furthermore, production of plant secondary metabolites, namely isoprenoids and stilbenes, has been demonstrated in this organism (Neves et al 2005;Katz et al 2011;Gaspar et al 2013;Song et al 2014). In addition, L. lactis is well known as an excellent host for the expression of both prokaryotic and eukaryotic proteins, and has been consequently used for development of live vaccine (Kunji et al 2005;Hernández et al 2007;Frelet-Barrand et al 2010;Pontes et al 2011).…”

Physiological Adaptation of the Bacterium Lactococcus lactis in Response to the Production of Human CFTR

“…Several cases have been reported in which functional overexpression of membrane proteins could be achieved in L. lactis, but not in E. coli (e.g. the human KDEL receptor, Na ϩ /tyrosine transporter (Tyt1) of Fusobacterium nucleatum and several membrane proteins from Arabidopsis) (5,(13)(14)(15)(16)(17). The use of L. lactis as host for (eukaryotic) membrane protein expression has been reviewed by Kunji et al (14).…”