Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins

Abstract

Background: Despite 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 findings: The 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/significance: Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.

Figure 1. Strategy used to transfer cDNA from Gateway entry vectors to L. lactis vectors.

A. Overview of the cloning procedure. In this strategy, recombination occurs between the attL sites from Gateway entry vectors (containing the cDNA coding for the proteins of interest) and the attR sites from a “destination” vector (att: attachment sites). This destination vector (pBS-RfA) is a derivative of pBlueScript which contains the reading frame cassette A (RfA cassette) surrounded by two EcoRV sites. By LR recombination, the first step thus generates a “shuttle” vector in which the gene of interest is surrounded by the two attB sites and two flanking EcoRV restriction sites. In order to generate blunt ends, the nisin inducible vector pNZ8148 is digested by NcoI and treated with the Klenow enzyme (pNZ8148NK). The cDNA excised from the “shuttle” vector pBS-RfA-cDNA with EcoRV (generating blunt ends) is ligated into the vector pNZ8148NK and placed under the control of the P_nisA promoter. Positive selection of recombinant vectors is obtained using digestion of the ligation products with NsiI. B. Resulting N- and C- termini of the ORF. Nucleotide sequences at each side of the ORF (upper panel: 5′ORF and lower panel: 3′ORF). In bold, the ATG (start codon) and TAG (stop codon) of the cDNA. CCATG in bold corresponds to the NcoI site after digestion and treatment with the Klenow enzyme; the ATG within the NcoI site is in the reading frame of the ATG of the cDNA. The attB sites are in italics and the two half-sites resulting from EcoRV digestion are in bold italics.

Figure 2. Expression of plant membrane proteins in L. lactis.

A. Impact of nisin concentration on the expression level of AtHMA1. Production of the AtHMA1 protein after induction by 1/1000^th, 1/200^th and 1/100^th dilution of nisin in culture medium. B. Production of the ceQORH and AtHMA1 proteins. C. Production of the AtAATP1 protein. D. Production of the four Arabidopsis P_1B-ATPases. Total membrane proteins (15 µg for panels A and D, 10 µg for panel B and 50 µg for panel C), were separated in a 10% SDS-PAGE and analyzed by western blot performed using an HRP conjugate specific to the Strep-tag II. Arrows indicate the positions of the expressed proteins. In panels B and C, expressed proteins contain an additional N-terminal sequence resulting from the translation of the attB sites. In panels A and D, c- means crude membrane proteins derived from bacteria containing the empty pNZ8148 vector. In panels B, C and D, defined amounts of a positive control protein (c+, Strep-tag II protein) were loaded to estimate the expression levels of the recombinant proteins.

Figure 3. Impact of the attB sites on the production level of membrane proteins in L. lactis.

A. Production of the ceQORH protein using the Gateway compatible (ceQORHatt) or the classical (ceQORHnh) cloning strategies. Control protein: 1 µg of ceQORH rec corresponding to the recombinant ceQORH protein produced in E. coli (Miras et al., 2002). B. AtHMA1 production using the Gateway compatible (AtHMA1att) or the classical (AtHMA1nh) cloning strategies. Control protein: 200 ng of the 28 kDa Strep-tag II protein. Total membrane proteins (5 µg for panel A and 15 µg for panel B) were separated in a 10% SDS-PAGE and analyzed by western blot using a polyclonal antibody raised against the ceQORH protein (panel A) or an HRP conjugate specific to the Strep-tag II (panel B). c-, crude membrane proteins derived from bacteria containing the empty pNZ8148 vector.

Figure 4. Solubilization and purification of the recombinant ceQORH protein produced in L. lactis.

A. Impact of salts, pH and detergents on the solubilization of the ceQORH protein from crude Lactococcus membrane proteins. Treatments of membrane proteins (1 µg/µL) with various concentrations of salt, detergent or NaOH are described in the Materials & methods section. Solubilized proteins (S) were separated from insoluble membrane proteins (I) by centrifugation. Proteins were analyzed by Coomassie blue-stained SDS-PAGE (upper panel) and by western blot (lower panel) performed using the anti-ceQORH antibody (lanes 1, 2, 4) or a HRP conjugate specific to the Strep-tag II (lanes 3, 5–10). B. Purification of the recombinant ceQORH protein on a Strep-Tactin Sepharose matrix. Membrane proteins solubilized with 1 M NaCl and desalted on a PD10 column (L) were loaded on the column containing the affinity matrix; F, flowthrough; W, washing fraction; E1 to E5, elution fractions. Aliquots (20 µL) of all fractions were loaded on a 12% SDS-PAGE further stained with Coomassie Blue. The arrow indicates the ceQORH protein.

Figure 5. Solubilization and purification of two intrinsic recombinant AtHMA1 and AtHMA6 proteins.

Panels A, B, D, E: Solubilization of AtHMA1 and AtHMA6 using detergents. Membrane proteins (MP, 4 µg/µL) were incubated in 50 mM Tris-HCl (pH 8.0), 100 mM NaCl and subsequently centrifuged to eliminate soluble proteins (W, washing). Membrane pellets were solubilized in the same buffer containing 1% DDM (w/v), 0.32% (w/v) C₁₂E₈ and 100 µM TCEP. After incubation for 1h30, solubilized membrane proteins (S) were separated from insoluble proteins (I) by centrifugation. Aliquots (15 µg of crude MP and 10 µL of resulting fractions W, I and S) were loaded on a 10% SDS-PAGE further stained with Coomassie blue (panels A, D) and by western blot (panels B, E) using the HRP conjugate specific to the Strep-tag II. Panels C and F: purification of AtHMA1 and AtHMA6, respectively, using a Strep-Tactin Sepharose matrix in a buffer containing 100 µM TCEP and 0.1% (w/v) DDM. Solubilized membrane proteins were loaded (L, 10 µL) on the column (F, 10 µL flowthrough). After washing the matrix (W, 10 µL), the bound proteins were eluted (E1 to E5, 20 µL) by addition of 2.5 mM desthiobiotin. Fractions were loaded on a 10% SDS-PAGE further stained with Coomassie blue. Arrows indicate the AtHMA1 (panels A, B, C) and the AtHMA6 (panels D, E, F) proteins.

Figure 6. Transport kinetics (ATP uptake) of the recombinant AtAATP1 protein in L. lactis.

After induction of expression with nisin for 4 h at 30°C, L. lactis cells expressing the AtAATP1 protein or L. lactis cells containing the empty vector (as a negative control), were incubated with 50 µM [α-³²P] ATP for the indicated time periods. Data are the mean of four independent experiments and standard error to the mean are displayed. The graph shows the difference of ATP transport efficiency (expressed in pmol ATP/µg total membrane proteins) between L. lactis cells expressing the AtAATP1 protein (full circles) or L. lactis cells containing the empty vector (empty circles).