Setup and Applications of Modular Protein Expression Toolboxes (MoPET) for Mammalian Systems.

The design and generation of an optimal protein expression construct is the first and essential step in the characterization of any protein of interest. However, the exchange and modification of the coding and/or noncoding elements to analyze their effect on protein function or generating the optimal result can be a tedious and time-consuming process using standard molecular biology cloning methods. To streamline the process to generate defined expression constructs or libraries of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed (Weber E, PloS One 12(5):e0176314, 2017). The system applies Golden Gate cloning as an assembly method and follows the standardized modular cloning (MoClo) principle (Weber E, PloS One 6(2):e16765, 2011). This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules effecting protein expression with a focus on minimizing the cloning burden in coding regions. The original MoPET system consists of 53 defined DNA modules divided into eight functional main classes and can be flexibly expanded dependent on the need of the experimenter and expression host. However, already with a limited set of only 53 modules, 792,000 different constructs can be rationally designed or used to generate combinatorial expression optimization libraries. We provide here a detailed protocol for the (1) design and generation of level 0 basic parts, (2) generation of defined expressions constructs, and (3) generation of combinatorial expression libraries.

Modular Protein Expression Toolbox (MoPET), a standardized assembly system for defined expression constructs and expression optimization libraries.

The design and generation of an optimal expression construct is the first and essential step in in the characterization of a protein of interest. Besides evaluation and optimization of process parameters (e.g. selection of the best expression host or cell line and optimal induction conditions and time points), the design of the expression construct itself has a major impact. However, the path to this final expression construct is often not straight forward and includes multiple learning cycles accompanied by design variations and retesting of construct variants, since multiple, functional DNA sequences of the expression vector backbone, either coding or non-coding, can have a major impact on expression yields. To streamline the generation of defined expression constructs of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed. This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules with a minimal cloning burden. Combining these features with a standardized cloning strategy facilitates the identification of optimized DNA expression constructs in shorter time. The MoPET system currently consists of 53 defined DNA modules divided into eight functional classes and can be flexibly expanded. However, already with the initial set of modules, 792,000 different constructs can be rationally designed and assembled. Furthermore, this starting set was used to generate small and mid-sized combinatorial expression optimization libraries. Applying this screening approach, variants with up to 60-fold expression improvement have been identified by MoPET variant library screening.

A library of synthetic transcription activator-like effector-activated promoters for coordinated orthogonal gene expression in plants.

A library of synthetic promoters containing the binding site of a single designer transcription activator-like effector (dTALE) was constructed. The promoters contain a constant sequence, consisting of an 18-base long dTALE-binding site and a TATA box, flanked by degenerate sequences of 49 bases downstream and 19 bases upstream. Forty-three of these promoters were sequenced and tested in transient assays in Nicotiana benthamiana using a GUS reporter gene. The strength of expression of the promoters ranged from around 5% to almost 100% of the viral 35S promoter activity. We then demonstrated the utility of these promoters for metabolic engineering by transiently expressing three genes for the production of a plant diterpenoid in N. benthamiana. The simplicity of the promoter structure shows great promise for the development of genetic circuits, with wide potential applications in plant synthetic biology and metabolic engineering.

Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system.

Recent progress in the field of synthetic biology has led to the creation of cells containing synthetic genomes. Although these first synthetic organisms contained copies of natural genomes, future work will be directed toward engineering of organisms with modified genomes and novel phenotypes. Much work, however, remains to be done to be able to routinely engineer novel biological functions. As a tool that will be useful for such purpose, we have recently developed a modular cloning system (MoClo) that allows high throughput assembly of multiple genetic elements. We present here new features of this cloning system that allow to increase the speed of assembly of multigene constructs. As an example, 68 DNA fragments encoding basic genetic elements were assembled using three one-pot cloning steps, resulting in a 50 kb construct containing 17 eukaryotic transcription units. This cloning system should be useful for generating the multiple construct variants that will be required for developing gene networks encoding novel functions, and fine-tuning the expression levels of the various genes involved.

Assembly of designer TAL effectors by Golden Gate cloning.

Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the target sequence. We present here a strategy for engineering of TALE proteins with novel DNA binding specificities based on the 17.5 repeat-containing AvrBs3 TALE as a scaffold. For each of the 17 full repeats, four module types were generated, each with a distinct base preference. Using this set of 68 repeat modules, recognition domains for any 17 nucleotide DNA target sequence of choice can be constructed by assembling selected modules in a defined linear order. Assembly is performed in two successive one-pot cloning steps using the Golden Gate cloning method that allows seamless fusion of multiple DNA fragments. Applying this strategy, we assembled designer TALEs with new target specificities and tested their function in vivo.

A modular cloning system for standardized assembly of multigene constructs.

The field of synthetic biology promises to revolutionize biotechnology through the design of organisms with novel phenotypes useful for medicine, agriculture and industry. However, a limiting factor is the ability of current methods to assemble complex DNA molecules encoding multiple genetic elements in various predefined arrangements. We present here a hierarchical modular cloning system that allows the creation at will and with high efficiency of any eukaryotic multigene construct, starting from libraries of defined and validated basic modules containing regulatory and coding sequences. This system is based on the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. We constructed a 33 kb DNA molecule containing 11 transcription units made from 44 individual basic modules in only three successive cloning steps. This modular cloning (MoClo) system can be readily automated and will be extremely useful for applications such as gene stacking and metabolic engineering.

Efficacy and safety of angiotensin II receptor type 1 antagonists in children and adolescents.

Our purpose was to evaluate the effects of angiotensin II receptor type 1 antagonists (ARAs) in children and adolescents with hypertension or/and several kinds of nephropathies on blood pressure (BP) and proteinuria and to evaluate related safety issues. Data sources were Medline, Embase, The Cochrane Library, BIOSIS Previews, contact with investigators and manufacturers, personal bibliography of the lead author, and manual searches. We selected randomized controlled trials (RCTs), uncontrolled trials, and case series investigating ARAs in children and adolescents, as well as case reports about adverse events and the embryotoxic effects of ARAs in children. In four RCTs with 698 individuals, mean systolic blood pressure (BP) decreased by 10.5 mmHg [95% confidence interval (CI) 9.8-11.2] and mean diastolic BP by 6.4 mmHg (95% CI 5.8-7.0). Proteinuria decreased by 30-64% (range) in two RCTs and four case series. Safety data were comparable with adult safety data. ARAs can be considered effective and safe in lowering BP and proteinuria in the pediatric age group. The correlation between the surrogate parameters BP and proteinuria with clinical endpoints is documented to a large degree. The evidence is based on RCTs and also on lower evidence levels, such as case series. In some conditions, RCTs in children are not feasible. Registers could provide more evidence in the future.

Refinement of the Xanthomonas campestris pv. vesicatoria hrpD and hrpE operon structure.

The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (T3S) system which is encoded in the 23-kb hypersensitive response and pathogenicity (hrp) gene cluster. The T3S system is essential for pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. In this study, we revisited the operon structure of the right part of the hrp gene cluster. Based on complementation experiments of transposon insertions and reverse-transcription polymerase chain reaction analyses, the hrpD operon contains hrcQ, hrcR, hrcS, and hpaA, whereas hrcD, hrpD6, and hrpE belong to the hrpE operon. We determined the transcriptional start site of the hrpE operon and showed that there is a promoter upstream of hrcD containing a plant-inducible promoter box. Conserved secondary mRNA structures in the intergenic region between hrpD6 and hrpE suggest a posttranscriptional regulated expression of hrpE. Based on comparisons of different hrp gene clusters and the analysis of evolutionary rates, we propose that the hrpE transcriptional unit was integrated into the hrp gene cluster at a later time.

Positive selection of the Hrp pilin HrpE of the plant pathogen Xanthomonas.

The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (TTS) system which is encoded by the 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. The TTS system is necessary for pathogenicity in susceptible hosts and induction of the hypersensitive response in resistant plants. At the cell surface, the TTS system is associated with an extracellular filamentous structure, the Hrp pilus, which serves as a conduit for the transfer of bacterial proteins into the plant cell cytosol. The major pilus component, the HrpE pilin, is unique to xanthomonads. Previous work showed that HrpE contains two regions: a hypervariable surface-exposed domain, including the N-terminal secretion signal, and a C-terminal polymerization domain. In this study, the evolutionary rate of the hrpE gene was analyzed. Twenty-one alleles were cloned, sequenced, and compared with five known hrpE alleles. The ratio of synonymous (K(s)) and nonsynonymous (K(a)) substitution rates shows that parts of the HrpE N terminus are subjected to positive selection and the C terminus is subjected to purifying selection. The trade-off between positive and purifying selection at the very-N terminus allowed us to ascertain the amphipathic alpha-helical nature of the TTS signal. This is the first report of a surface structure from a plant-pathogenic bacterium that evolved under the constraint of positive selection and hints to the evolutionary adaptation of this extracellular appendage to avoid recognition by the plant defense surveillance system.

Targeting of two effector protein classes to the type III secretion system by a HpaC- and HpaB-dependent protein complex from Xanthomonas campestris pv. vesicatoria.

The Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria translocates effector proteins via a specialized type III secretion (TTS) system into the host cell cytosol. The efficient secretion of many effector proteins depends on the global TTS chaperone HpaB. Here, we identified a novel export control protein, HpaC, which significantly contributes to bacterial pathogenicity. Deletion of hpaC leads to a severe reduction in secretion of effector proteins and the putative type III translocon proteins HrpF and XopA. By contrast, secretion of the TTS pilus protein HrpE is not affected. We provide experimental evidence that HpaC differentiates between two classes of effector proteins. Using an in vivo reporter assay, we found that HpaC specifically promotes the translocation of the effector proteins XopJ and XopF1 into the plant cell, whereas AvrBs3 and XopC are efficiently translocated even in the absence of HpaC. Similar findings were obtained for HpaB. Inhibition of protein synthesis suggests that HpaB is involved in the secretion of stored effector proteins. Furthermore, protein-protein interaction studies revealed that HpaB and HpaC form an oligomeric protein complex and that they interact with members of both effector protein classes and the conserved TTS system component HrcV. Taken together, our data indicate that HpaB and HpaC play a central role in recruiting TTS substrates to the secretion apparatus.

Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence.

The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.

Domain structure of HrpE, the Hrp pilus subunit of Xanthomonas campestris pv. vesicatoria.

The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (TTS) system necessary for pathogenicity in susceptible hosts and induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. X. campestris pv. vesicatoria produces filamentous structures, Hrp pili, at the cell surface under hrp-inducing conditions. The Hrp pilus acts as a cell surface appendage of the TTS system and serves as a conduit for the transfer of bacterial effector proteins into the plant cell cytosol. The major pilus component, the HrpE pilin, is unique to xanthomonads and is encoded within the hrp gene cluster. In this study, functional domains of HrpE were mapped by linker-scanning mutagenesis and by reporter protein fusions to an N-terminally truncated avirulence protein (AvrBs3Delta2). Thirteen five-amino-acid peptide insertion mutants were obtained and could be grouped into six phenotypic classes. Three permissive mutations were mapped in the N-terminal half of HrpE, which is weakly conserved within the HrpE protein family. Four dominant-negative peptide insertions in the strongly conserved C-terminal region suggest that this domain is critical for oligomerization of the pilus subunits. Reporter protein fusions revealed that the N-terminal 17 amino acid residues act as an efficient TTS signal. From these results, we postulate a three-domain structure of HrpE with an N-terminal secretion signal, a surface-exposed variable region of the N-terminal half, and a C-terminal polymerization domain. Comparisons with a mutant study of HrpA, the Hrp pilin from Pseudomonas syringae pv. tomato DC3000, and hydrophobicity plot analyses of several nonhomologous Hrp pilins suggest a common architecture of Hrp pilins of different plant-pathogenic bacteria.

Effects of protein and gene transfer of the angiopoietin-1 fibrinogen-like receptor-binding domain on endothelial and vessel organization.

The vessel-stabilizing effect of angiopoietin-1 (Ang1)/Tie2 receptor signaling is a potential target for pro-angiogenic therapies as well as anti-angiogenic inhibition of tumor growth. We explored the endothelial and vascular specific activities of the Ang1 monomer, i.e. dissociated from its state as an oligomer. A truncated monomeric Ang1 variant (i.e. DeltaAng1) containing the isolated fibrinogen-like receptor-binding domain of Ang1 was created and recombinantly produced in insect cells. DeltaAng1 ligated the Tie2 receptor without triggering its phosphorylation. Moreover, monomeric DeltaAng1 was observed to bind alpha(5)beta(1) integrin with similar affinity compared with Tie2. Unexpectedly, in vitro treatment of endothelial cells with DeltaAng1 showed some of the known effects of full-length Ang1, including inhibition of basal endothelial cell permeability and stimulation of cell adhesion as well as activation of MAPKs. Local treatment of the microvasculature of the developing chicken chorioallantoic membrane with the DeltaAng1 protein led to profound reduction of the mean vascular length density, thinning of vessels, and reduction of the number of vessel branching points. Similar effects were observed in side-by-side experiments with the recombinant full-length Ang1 protein. These effects of simplification of the vessel branching pattern were confirmed through local gene transfer with lentiviral particles encoding DeltaAng1 or full-length Ang1. Together, our findings suggest a potential use for exogenous Ang1 in reducing rather than increasing vascular density. Furthermore, we show that the isolated receptor-binding domain of Ang1 is capable of mediating some effects of full-length Ang1 independently of Tie2 phosphorylation, possibly through integrin ligation.

The type III-dependent Hrp pilus is required for productive interaction of Xanthomonas campestris pv. vesicatoria with pepper host plants.

The plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria expresses a type III secretion system that is necessary for both pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Here we show that X. campestris pv. vesicatoria produces filamentous structures, the Hrp pili, at the cell surface under hrp-inducing conditions. Analysis of purified Hrp pili and immunoelectron microscopy revealed that the major component of the Hrp pilus is the HrpE protein which is encoded in the hrp gene cluster. Sequence homologues of hrpE are only found in other xanthomonads. However, hrpE is syntenic to the hrpY gene from another plant pathogen, Ralstonia solanacearum. Bioinformatic analyses suggest that all major Hrp pilus subunits from gram-negative plant pathogens may share the same structural organization, i.e., a predominant alpha-helical structure. Analysis of nonpolar mutants in hrpE demonstrated that the Hrp pilus is essential for the productive interaction of X. campestris pv. vesicatoria with pepper host plants. Furthermore, a functional Hrp pilus is required for type III-dependent protein secretion. Immunoelectron microscopy revealed that type III-secreted proteins, such as HrpF and AvrBs3, are in close contact with the Hrp pilus during and/or after their secretion. By systematic analysis of nonpolar hrp/hrc (hrp conserved) and hpa (hrp associated) mutants, we found that Hpa proteins as well as the translocon protein HrpF are dispensable for pilus assembly, while all other Hrp and Hrc proteins are required. Hence, there are no other conserved Hrp or Hrc proteins that act downstream of HrpE during type III-dependent protein translocation.

Venous sampling can be crucial in identifying the testicular origin of idiopathic male luteinising hormone-independent sexual precocity.

UNLABELLED: It has been recently shown that male LH-independent sexual precocity is caused by a somatic activating mutation in the luteinising hormone receptor (LHR) of Leydig cell tumours. In each of the patients described to date, the tumour was a well-defined, single encapsulated nodule. We present a 5.7-year-old boy with nodular Leydig cell hyperplasia, who harbours a somatic mutation of the LHRgene. The boy showed the clinical features of severe sexual precocity caused by LH-independent testosterone hypersecretion. Congenital adrenal hyperplasia, hCG- or androgen-secreting tumours, McCune-Albright syndrome, and familial male-limited precocious puberty (or testotoxicosis) were all ruled out as possible causes. A hypoechoic area was detected at the cranial pole of his right testis and a biopsy was performed. Histological examination revealed a lack of mature Leydig cells. When DNA from the affected tissue was isolated and sequenced, no somatic mutation of the LHR gene was found. To further determine the origin of the elevated testosterone levels, venous sampling was performed. Blood samples taken from the right spermatic vein showed an elevated serum testosterone concentration of 259 nmol/l. Unilateral orchiectomy of the right testis was performed, and systemic testosterone concentrations normalised. Histological examination revealed nodular Leydig cell hyperplasia. DNA analysis of the nodular tissue showed a heterozygous mutation in exon 11 of the LHR gene, which caused the replacement of aspartic acid at codon 578 by histidine. CONCLUSION: the somatic activating mutation (Asp578His) of the luteinising hormone receptor gene is not only present in Leydig cell adenomas, but can also be found in nodular Leydig cell hyperplasia. Venous sampling can play a vital role in determining the origin of elevated testosterone levels.