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Among the small number of expression systems validated for the mass production of eukaryotic membrane proteins (EMPs), the methylotrophic yeast Pichia pastoris stands as one of the most efficient hosts. This system has been used to produce crystallization-grade proteins for a variety of EMPs, from which high-resolution 3D structures have been determined. This unit describes a set of guidelines and instructions to overexpress membrane proteins using the P. pastoris system. Using a G protein-coupled receptor (GPCR) as a model EMP, these protocols illustrate the necessary steps, starting with the design of the DNA sequence to be expressed, through the preparation and analysis of samples containing the corresponding membrane protein of interest. In addition, recommendations are given on a series of experimental parameters that can be optimized to substantially improve the amount and/or the functionality of the expressed EMPs.

When planning any heterologous expression experiment, the very first critical step is related to the design of the overall strategy, hence to the selection of the most adapted expression vector. The very flexible Pichia pastoris system offers a broad range of possibilities for the production of secreted, endogenous or membrane proteins thanks to a combination of various plasmid backbones, selection markers, promoters and fusion sequences introduced into dedicated host strains. The present chapter provides some guidelines on the choice of expression vectors and expression strategies. It also brings the reader a complete toolbox from which plasmids and fusion sequences can be picked and assembled to set up appropriate expression vectors. Finally, it provides standard starting protocols for the preparation of the selected plasmids and their use for host strain transformation.

Thioredoxins (Trxs) h, small disulfide reductases, and NADP-thioredoxin reductases (NTRs) have been shown to accumulate in seeds of different plant species and play important roles in seed physiology. However, little is known about the identity, properties, and subcellular location of Trx h isoforms that are abundant in legume seeds. To fill this gap, in this work, we characterized the Trx h family of Medicago truncatula, a model legume, and then explored the activity and localization of Trx h isoforms accumulating in seeds. Twelve Trx h isoforms were identified in M. truncatula. They belong to the groups previously described: h1 to h3 (group I), h4 to h7 (group II), and h8 to h12 (group III). Isoforms of groups I and II were found to be reduced by M. truncatula NTRA, but with different efficiencies, Trxs of group II being more efficiently reduced than Trxs of group I. In contrast, their insulin disulfide-reducing activity varies greatly and independently of the group to which they belong. Furthermore, Trxs h1, h2, and h6 were found to be present in dry and germinating seeds. Trxs h1 and, to a lesser extent, h2 are abundant in both embryonic axes and cotyledons, while Trx h6 is mainly present in cotyledons. Thus, M. truncatula seeds contain distinct isoforms of Trx h that differ in spatial distribution and kinetic properties, suggesting that they play different roles. Because we show that Trx h6 is targeted to the tonoplast, the possible role of this isoform during germination is finally discussed.

G-protein-coupled receptors (GPCRs), the largest family of membrane proteins, represent ideal therapeutic targets for a number of disorders and diseases. Besides cell-based assays and high throughput screening (HTS), and thanks to the availability of milligram quantities of active purified receptors, protein-based approaches focusing on soluble GPCRs are growingly being used in this drug discovery effort. Along with the exploitation of GPCRs structures, innovative biochemical and biophysical approaches open up new routes for improving the knowledge of structure-activity relationships, for the identification of novel interacting partners and for the determination of receptor behaviour in different model environments. This review summarizes the state-of-the-art methodologies that robustly allow for the production and purification of soluble and active GPCRs, as well as the main outcomes that have been recently gained in GPCR biology using a panel of such protein-based approaches.

The turn of the century welcomed major developments in redox biology. In plants, proteomics made possible the identification of proteins linked to thioredoxin (Trx), initially in chloroplasts and then other cell compartments. Two procedures, one based on thiol specific probes and the other on mutant Trx proteins, facilitated the labeling or isolation of potential Trx targets that were later identified with proteomic approaches. As a result, the number of targets in land plants increased 10-fold from fewer than 40 to more than 400. Additional targets have been identified in green algae and cyanobacteria, making a grand total of 500 in oxygenic photosynthetic organisms. Collectively these proteins have the potential to influence virtually every major process of the cell. A number of laboratories currently seek to confirm newly identified Trx targets by biochemical and genetic approaches. Almost certainly many new targets become redox active during oxidative stress, enabling the plant to cope with changing environments. Under these conditions, certain targets may be glutathionylated or nitrosylated such that reversion to the original reduced state is facilitated not only by Trx, but also, in some cases preferably, by glutaredoxin. When judging changes linked to Trx, it is prudent to recognize that effects transcend classical light/dark or oxidative regulation and fall in other arenas, in some cases yet to be defined. While future work will continue to give insight into functional details, it is clear that Trx plays a fundamental role in regulating diverse processes of the living cell.

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.

Thioredoxins (Trxs) constitute a family of small proteins in plants. This family has been extensively characterized in Arabidopsis (Arabidopsis thaliana), which contains six different Trx types: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A detailed study of this family in the model legume Medicago truncatula, realized here, has established the existence of two isoforms that do not belong to any of the types previously described. As no possible orthologs were further found in either rice (Oryza sativa) or poplar (Populus spp.), these novel isoforms may be specific for legumes. Nevertheless, on the basis of protein sequence and gene structure, they are both related to Trxs m and probably have evolved from Trxs m after the divergence of the higher plant families. They have redox potential values similar to those of the classical Trxs, and one of them can act as a substrate for the M. truncatula NADP-Trx reductase A. However, they differ from classical Trxs in that they possess an atypical putative catalytic site and lack disulfide reductase activity with insulin. Another important feature is the presence in both proteins of an N-terminal extension containing a putative signal peptide that targets them to the endoplasmic reticulum, as demonstrated by their transient expression in fusion with the green fluorescent protein in M. truncatula or Nicotiana benthamiana leaves. According to their pattern of expression, these novel isoforms function specifically in symbiotic interactions in legumes. They were therefore given the name of Trxs s, s for symbiosis.

Germination of cereals is accompanied by extensive change in the redox state of seed proteins. Proteins present in oxidized form in dry seeds are converted to the reduced state following imbibition. Thioredoxin (Trx) appears to play a role in this transition in cereals. It is not known, however, whether Trx-linked redox changes are restricted to cereals or whether they take place more broadly in germinating seeds. To gain information on this point, we have investigated a model legume, Medicago truncatula. Two complementary gel-based proteomic approaches were followed to identify Trx targets in seeds: Proteins were (1) labeled with a thiol-specific probe, monobromobimane (mBBr), following in vitro reduction by an NADP/Trx system, or (2) isolated on a mutant Trx affinity column. Altogether, 111 Trx-linked proteins were identified with few differences between axes and cotyledons. Fifty nine were new, 34 found previously in cereal or peanut seeds, and 18 in other plants or photosynthetic organisms. In parallel, the redox state of proteins assessed in germinating seeds using mBBr revealed that a substantial number of proteins that are oxidized or partly reduced in dry seeds became more reduced upon germination. The patterns were similar for proteins reduced in vivo during germination or in vitro by Trx. In contrast, glutathione and glutaredoxin were less effective as reductants in vitro. Overall, more than half of the potential targets identified with the mBBr labeling procedure were reduced during germination. The results provide evidence that Trx functions in the germination of seeds of dicotyledons as well as monocotyledons.

The NADPH/NADP-thioredoxin (Trx) reductase (NTR)/Trx system (NTS) is a redox system that plays a posttranslational regulatory role by reducing protein targets involved in crucial cellular processes in microorganisms and animals. In plants, the system includes several h type Trx isoforms and has been shown to intervene in reserve mobilization during early seedling growth of cereals. To determine whether NTS was operational during germination of legume seeds and which Trx h isoforms could be implicated, Trx h isoforms expression was monitored in germinating pea (Pisum sativum cv Baccara) seeds, together with the amount of NTR and NADPH. Two new isoforms were identified: Trx h3, similar to the two isoforms already described in pea but not expressed in seeds; and the more divergent isoform, Trx h4. Active recombinant proteins were produced in Escherichia coli and used to raise specific antibodies. The expression of new isoforms was analyzed at both mRNA and protein levels. The lack of correlation between mRNA and protein abundances suggests the occurrence of posttranscriptional regulation. Trx h3 protein amount remained constant in both axes and cotyledons of dry and imbibed seeds but then decreased 2 d after radicle protrusion. In contrast, Trx h4 was only expressed in axes of dry and imbibed seeds but not in germinated seeds or in seedlings, therefore appearing as closely linked to germination. The presence of NTR and NADPH in seeds suggests that NTS could be functional during germination. The possible role of Trx h3 and h4 in this context is discussed.