Divergent effects of itaconate isomers on Coxiella burnetii growth in macrophages and in axenic culture.

Aconitate decarboxylase-1 (ACOD1) is expressed by activated macrophages and generates itaconate that exerts anti-microbial and immunoregulatory effects. ACOD1-itaconate is essential for macrophage-mediated control of the intracellular pathogen Coxiella (C.) burnetii, which causes Q fever. Two isomers of itaconate, mesaconate and citraconate, have overlapping yet distinct activity on macrophage metabolism and inflammatory gene expression. Here, we found that all three isomers inhibited the growth of C. burnetii in axenic culture in ACCM-2 medium. However, only itaconate reduced C. burnetii replication efficiently in Acod1-/- macrophages. In contrast, addition of citraconate strongly increased C. burnetii replication in Acod1+/- macrophages, whereas mesaconate weakly enhanced bacterial burden in Acod1-/- macrophages. Analysis of intracellular isomers showed that exogenous citraconate and mesaconate inhibited the generation of itaconate by infected Acod1+/- macrophages. Uptake of added isomers into Acod1-/- macrophages was increased after infection for itaconate and mesaconate, but not for citraconate. Mesaconate, but not citraconate, competed with itaconate for uptake into macrophages. Taken together, inhibition of itaconate generation by macrophages and interference with the uptake of extracellular itaconate could be identified as potential mechanisms behind the divergent effects of citraconate and mesaconate on C. burnetii replication in macrophages or in axenic culture.

Amino acid positions near the active site determine the reduced activity of human ACOD1 compared to murine ACOD1.

cis-Aconitate decarboxylase (ACOD1, IRG1) converts cis-aconitate to the immunomodulatory and antibacterial metabolite itaconate. Although the active site residues of human and mouse ACOD1 are identical, the mouse enzyme is about fivefold more active. Aiming to identify the cause of this difference, we mutated positions near the active site in human ACOD1 to the corresponding residues of mouse ACOD1 and measured resulting activities in vitro and in transfected cells. Interestingly, Homo sapiens is the only species with methionine instead of isoleucine at residue 154 and introduction of isoleucine at this position increased the activity of human ACOD1 1.5-fold in transfected cells and 3.5-fold in vitro. Enzyme activity of gorilla ACOD1, which is almost identical to the human enzyme but has isoleucine at residue 154, was similar to the mouse enzyme in vitro. Met154 in human ACOD1 forms a sulfur-π bond to Phe381, which is positioned to impede access of the substrate to the active site. It appears that the ACOD1 sequence has changed at position 154 during human evolution, resulting in a pronounced decrease in activity. This change might have offered a selective advantage in diseases such as cancer.

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-ß Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.

ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.

Crystal structure of cis-aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis.

cis-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by Aspergillus terreus We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from A. terreus Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas Aspergillus CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and Aspergillus We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid.

ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.

Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNß. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNß-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.

Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.

A method for specifically targeting two independent genomic integration sites for co-expression of genes in CHO cells.

Stable mammalian production cell lines in suspension culture enable the reproducible expression of target genes in any desired scale using bioreactor technology. Targeted integration methods have been developed to cut down timelines for the generation of stable producer cell lines. The powerful Flp recombinase mediated cassette exchange (RMCE) technique allows fast integration of target genes in preselected and optimized high expression loci in so called master cell lines. Up to now, these cells only enable the expression from a single locus. Here, we describe the set-up required for the generation of multiple tagged master cell lines on the example of a binary RMCE expression system in the glycosylation mutant CHO Lec3.2.8.1 cell line. We show how this technology is used for the expression of proteins from multiple loci by generating a binary RMCE expression system. The tools and strategy for the construction of binary master cell lines with different combinations of expression level are described in detail. The binary production cell lines show independent expression of the individual exchange loci of the producer cell lines. The expression level for the model protein tdTomato is the cumulative expression for the chosen combination of the expression loci of the master cell line. This binary RMCE expression system can be further developed to a multi RMCE expression system for co-expression of protein complex subunits with predetermined expression ratio of each individual exchange locus.

Stable mammalian producer cell lines for structural biology.

The mammalian cell lines HEK293 and CHO have become important expression hosts in structural biology. Generating stable mammalian cell lines remains essential for studying the function and structure of recombinant proteins, despite the emergence of highly efficient transient transfection protocols. Production with stable cell lines can be scaled up easily and high volumetric product yield can be achieved. Protein structure reports of the past two years that used stable cell lines were surveyed for this review. Well-established techniques and novel approaches for generating stable cell lines and stable cell pools are presented, including cell sorting, site-specific recombination, transposons, the Lentivirus system and phage integrases. Host cell line optimization by endoglycosidase overexpression and sequence-specific genome engineering is highlighted.

High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells.

BACKGROUND: The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. RESULTS: In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. CONCLUSION: Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.

Structural characterization of amorfrutins bound to the peroxisome proliferator-activated receptor γ.

Amorfrutins are a family of natural products with high affinity to the peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor regulating lipid and glucose metabolism. The PPARγ agonist rosiglitazone increases insulin sensitivity and is effective against type II diabetes but has severe adverse effects including weight gain. Amorfrutins improve insulin sensitivity and dyslipidemia but do not enhance undesired fat storage. They bear potential as therapeutics or prophylactic dietary supplements. We identified amorfrutin B as a novel partial agonist of PPARγ with a considerably higher affinity than that of previously reported amorfrutins, similar to that of rosiglitazone. Crystal structures reveal the geranyl side chain of amorfrutin B as the cause of its particularly high affinity. Typical for partial agonists, amorfrutins 1, 2, and B bind helix H3 and the ß-sheet of PPARγ but not helix H12.

Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx.

BACKGROUND: The family of lysosome-associated membrane proteins (LAMP) comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68). LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of the glycoconjugate coat present on the inside of the lysosomal membrane, the 'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain of 150 to 200 amino acids with two disulfide bonds. RESULTS: The crystal structure of the conserved domain of human DC-LAMP was solved. It is the first high-resolution structure of a heavily glycosylated lysosomal membrane protein. The structure represents a novel ß-prism fold formed by two ß-sheets bent by ß-bulges and connected by a disulfide bond. Flexible loops and a hydrophobic pocket represent possible sites of molecular interaction. Computational models of the glycosylated luminal regions of LAMP-1 and LAMP-2 indicate that the proteins adopt a compact conformation in close proximity to the lysosomal membrane. The models correspond to the thickness of the lysosomal glycoprotein coat of only 5 to 12 nm, according to electron microscopy. CONCLUSION: The conserved luminal domain of lysosome-associated membrane proteins forms a previously unknown ß-prism fold. Insights into the structure of the lysosomal glycoprotein coat were obtained by computational models of the LAMP-1 and LAMP-2 luminal regions.

Amorfrutins are potent antidiabetic dietary natural products.

Given worldwide increases in the incidence of obesity and type 2 diabetes, new strategies for preventing and treating metabolic diseases are needed. The nuclear receptor PPARγ (peroxisome proliferator-activated receptor gamma) plays a central role in lipid and glucose metabolism; however, current PPARγ-targeting drugs are characterized by undesirable side effects. Natural products from edible biomaterial provide a structurally diverse resource to alleviate complex disorders via tailored nutritional intervention. We identified a family of natural products, the amorfrutins, from edible parts of two legumes, Glycyrrhiza foetida and Amorpha fruticosa, as structurally new and powerful antidiabetics with unprecedented effects for a dietary molecule. Amorfrutins bind to and activate PPARγ, which results in selective gene expression and physiological profiles markedly different from activation by current synthetic PPARγ drugs. In diet-induced obese and db/db mice, amorfrutin treatment strongly improves insulin resistance and other metabolic and inflammatory parameters without concomitant increase of fat storage or other unwanted side effects such as hepatoxicity. These results show that selective PPARγ-activation by diet-derived ligands may constitute a promising approach to combat metabolic disease.

Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.

Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).

Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli.

Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.

Impaired peripheral Th1 CD4+ T cell response to Escherichia coli proteins in patients with Crohn's disease and ankylosing spondylitis.

BACKGROUND: To clarify the impact of T cell responses towards enteric antigens for chronic intestinal inflammation, we determined T helper 1 reactivity towards conserved Escherichia coli proteins in patients with Crohn's disease (CD) and healthy individuals and patients with ankylosing spondylitis (AS), who also often show microscopic inflammatory lesions within the gut or even develop overt inflammatory bowel disease. METHODS: We determined the frequency of IFNγ+CD40L+ cells/CD4+ T cells after stimulation of whole blood with pools of E. coli proteins. RESULTS: The E. coli-specific Th1 response was significantly reduced in CD patients and to a lower extent also in AS patients. CONCLUSIONS: E. coli is a target for polyclonal Th1 responses in healthy individuals. The impairment of these responses in CD and AS patients might be due to recruitment of enterobacteria-specific Th1 cells to the gut or might reflect inadequate priming of adaptive immune response.

Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting.

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N-linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence-activated cell sorting (FACS) and site-specific gene excision to establish high-yield glycoprotein expression for structural studies with stable clones derived from the well-established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single-chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome-associated membrane protein 3 (LAMP3d). In both cases, stable GFP-expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.

Structural and functional characterization of human Iba proteins.

Iba2 is a homolog of ionized calcium-binding adapter molecule 1 (Iba1), a 17-kDa protein that binds and cross-links filamentous actin (F-actin) and localizes to membrane ruffles and phagocytic cups. Here, we present the crystal structure of human Iba2 and its homodimerization properties, F-actin cross-linking activity, cellular localization and recruitment upon bacterial invasion in comparison with Iba1. The Iba2 structure comprises two central EF-hand motifs lacking bound Ca2+. Iba2 crystallized as a homodimer stabilized by a disulfide bridge and zinc ions. Analytical ultracentrifugation revealed a different mode of dimerization under reducing conditions that was independent of Ca2+. Furthermore, no binding of Ca2+ up to 0.1 mM was detected by equilibrium dialysis. Correspondingly, Iba EF-hand motifs lack residues essential for strong Ca2+ coordination. Sedimentation experiments and microscopy detected pronounced, indistinguishable F-actin binding and cross-linking activity of Iba1 and Iba2 with induction of F-actin bundles. Fluorescent Iba fusion proteins were expressed in HeLa cells and co-localized with F-actin. Iba1 was recruited into cellular projections to a larger extent than Iba2. Additionally, we studied Iba recruitment in a Shigella invasion model that induces cytoskeletal rearrangements. Both proteins were recruited into the bacterial invasion zone and Iba1 was again concentrated slightly higher in the cellular extensions.

High throughput cloning with restriction enzymes.

The systematic structural analysis of many target proteins involves generating expression clones in high throughput. This requires robust laboratory procedures and benefits from laboratory automation and data management systems. This chapter gives an overview of the Protein Structure Factory, a structural genomics project focusing on human proteins, and presents the authors' method for cloning bacterial expression clones with the restriction enzymes BamHI and NotI and compatible enzymes. PCR amplification, product purification and digestion and vector ligation were adapted to the 96-well microtiter plate format.