MCCC2 is a novel mediator between mitochondria and telomere and functions as an oncogene in colorectal cancer.

BACKGROUND: The mitochondrial gene MCCC2, a subunit of the heterodimer of 3-methylcrotonyl-CoA carboxylase, plays a pivotal role in catabolism of leucine and isovaleric acid. The molecular mechanisms and prognostic value still need to be explored in the context of specific cancers, including colorectal cancer (CRC). METHODS: In vitro and in vivo cell-based assays were performed to explore the role of MCCC2 in CRC cell proliferation, invasion, and migration. Mitochondrial morphology, membrane potential, intracellular reactive oxygen species (ROS), telomerase activity, and telomere length were examined and analyzed accordingly. Protein complex formation was detected by co-immunoprecipitation (CO-IP). Mitochondrial morphology was observed by transmission electron microscopy (TEM). The Cancer Genome Atlas (TCGA) CRC cohort analysis, qRT-PCR, and immunohistochemistry (IHC) were used to examine the MCCC2 expression level. The association between MCCC2 expression and various clinical characteristics was analyzed by chi-square tests. CRC patients' overall survival (OS) was analyzed by Kaplan-Meier analysis. RESULTS: Ectopic overexpression of MCCC2 promoted cell proliferation, invasion, and migration, while MCCC2 knockdown (KD) or knockout (KO) inhibited cell proliferation, invasion, and migration. MCCC2 KD or KO resulted in reduced mitochondria numbers, but did not affect the gross ATP production in the cells. Mitochondrial fusion markers MFN1, MFN2, and OPA1 were all upregulated in MCCC2 KD or KO cells, which is in line with a phenomenon of more prominent mitochondrial fusion. Interestingly, telomere lengths of MCCC2 KD or KO cells were reduced more than control cells. Furthermore, we found that MCCC2 could specifically form a complex with telomere binding protein TRF2, and MCCC2 KD or KO did not affect the expression or activity of telomerase reverse transcriptase (TERT). Finally, MCCC2 expression was heightened in CRC, and patients with higher MCCC2 expression had favorable prognosis. CONCLUSIONS: Together, we identified MCCC2 as a novel mediator between mitochondria and telomeres, and provided an additional biomarker for CRC stratification.

Tracing the evolving dynamics and research hotspots of microbiota and immune microenvironment from the past to the new era.

Gut microbiota can regulate many physiological processes within gastrointestinal tract and other distal sites. Dysbiosis may not only influence chronic diseases like the inflammatory bowel disease (IBD), metabolic disease, tumor and its therapeutic efficacy, but also deteriorate acute injuries. This article aims to review the documents in this field and summarize the research hotspots as well as developing processes. Gut microbiota and immune microenvironment-related documents from 1976 to 2022 were obtained from the Web of Science Core Collection database. Bibliometrics was used to assess the core authors and journals, most contributive countries and affiliations together with hotspots in this field and keyword co-occurrence analysis. Data were visualized to help comprehension. Nine hundred and twelve documents about gut microbiota and immune microenvironment were retrieved, and the annual publications increased gradually. The most productive author, country, and affiliation were "Zitvogel L," USA and "UNIV TEXAS MD ANDERSON CANC CTR," respectively. FRONTIERS IN IMMUNOLOGY, CANCERS, and INTERNATIONAL JOURNAL OF MOLECULAR SCIENCE were the periodicals with most publications. Keyword co-occurrence analysis identified three clusters, including gut microbiota, inflammation, and IBD. Combined with the visualized analysis of documents and keyword co-occurrence as well as literature reading, we recognized three key topics of gut microbiota: cancer and therapy; immunity, inflammation and IBD; acute injuries and metabolic diseases. This article revealed researches on gut microbiota and immune microenvironment were growing. More attention should be given to the latest hotspots like gut microbiota, inflammation, IBD, cancer and immunotherapy, acute traumas, and metabolic diseases.IMPORTANCEGut microbiota can regulate many physiological processes within gastrointestinal tract and other distal sites. Dysbiosis may not only influence chronic diseases like inflammatory bowel disease (IBD), metabolic disease, tumor and its therapeutic efficacy, but also deteriorate acute injuries. While the application of bibliometrics in the field of gut microbiota and immune microenvironment still remains blank, which focused more on the regulation of the gut microbiota on the immune microenvironment of different kinds of diseases. Here, we intended to review and summarize the presented documents in gut microbiota and immune microenvironment field by bibliometrics. And we revealed researches on gut microbiota and immune microenvironment were growing. More attention should be given to the latest hotspots like gut microbiota, inflammation, IBD, cancer and immunotherapy, acute traumas, and metabolic diseases.

Mapping intellectual structure and research hotspots in the field of fibroblast-associated DFUs: a bibliometric analysis.

Background: Diabetic foot ulcers (DFUs) are one of the most popular and severe complications of diabetes. The persistent non-healing of DFUs may eventually contribute to severe complications such as amputation, which presents patients with significant physical and psychological challenges. Fibroblasts are critical cells in wound healing and perform essential roles in all phases of wound healing. In diabetic foot patients, the disruption of fibroblast function exacerbates the non-healing of the wound. This study aimed to summarize the hotspots and evaluate the global research trends on fibroblast-related DFUs through bibliometric analysis. Methods: Scientific publications on the study of fibroblast-related DFUs from January 1, 2000 to April 27, 2022 were retrieved from the Web of Science Core Collection (WoSCC). Biblioshiny software was primarily performed for the visual analysis of the literature, CiteSpace software and VOSviewer software were used to validate the results. Results: A total of 479 articles on fibroblast-related DFUs were retrieved. The most published countries, institutions, journals, and authors in this field were the USA, The Chinese University of Hong Kong, Wound Repair and Regeneration, and Seung-Kyu Han. In addition, keyword co-occurrence networks, historical direct citation networks, thematic map, and the trend topics map summarize the research hotspots and trends in this field. Conclusion: Current studies indicated that research on fibroblast-related DFUs is attracting increasing concern and have clinical implications. The cellular and molecular mechanisms of the DFU pathophysiological process, the molecular mechanisms and therapeutic targets associated with DFUs angiogenesis, and the measures to promote DFUs wound healing are three worthy research hotspots in this field.

Sequencing technology as a major impetus in the advancement of studies into rheumatism: A bibliometric study.

Background: Rheumatism covers a wide range of diseases with complex clinical manifestations and places a tremendous burden on humans. For many years, our understanding of rheumatism was seriously hindered by technology constraints. However, the increasing application and rapid advancement of sequencing technology in the past decades have enabled us to study rheumatism with greater accuracy and in more depth. Sequencing technology has made huge contributions to the field and is now an indispensable component and powerful tool in the study of rheumatism. Methods: Articles on sequencing and rheumatism, published from 1 January 2000 to 25 April 2022, were retrieved from the Web of Science™ (Clarivate™, Philadelphia, PA, USA) database. Bibliometrix, the open-source tool, was used for the analysis of publication years, countries, authors, sources, citations, keywords, and co-words. Results: The 1,374 articles retrieved came from 62 countries and 350 institutions, with a general increase in article numbers during the last 22 years. The leading countries in terms of publication numbers and active cooperation with other countries were the USA and China. The most prolific authors and most popular documents were identified to establish the historiography of the field. Popular and emerging research topics were assessed by keywords and co-occurrence analysis. Immunological and pathological process in rheumatism, classification, risks and susceptibility, and biomarkers for diagnosis were among the hottest themes for research. Conclusions: Sequencing technology has been widely applied in the study of rheumatism and propells research in the area of discovering novel biomarkers, related gene patterns and physiopathology. We suggest that further efforts be made to advance the study of genetic patterns related to rheumatic susceptibility, pathogenesis, classification and disease activity, and novel biomarkers.

A bibliometric analysis of the role of microbiota in trauma.

Introduction: Over the last several decades, the gut microbiota has been implicated in the formation and stabilization of health, as well as the development of disease. With basic and clinical experiments, scholars are gradually understanding the important role of gut microbiota in trauma, which may offer novel ideas of treatment for trauma patients. In this study, we purposed to summarize the current state and access future trends in gut microbiota and trauma research. Methods: We retrieved relevant documents and their published information from the Web of Science Core Collection (WoSCC). Bibliometrix package was responsible for the visualized analysis. Results: Totally, 625 documents were collected and the number of annual publications kept increasing, especially from 2016. China published the most documents while the USA had the highest local citations. The University of Colorado and Food & Function are respectively the top productive institution and journal, as PLOS One is the most local cited journal. With the maximum number of articles and local citations, Deitch EA is supported to be the most contributive author. Combining visualized analysis of keywords and documents and literature reading, we recognized two key topics: bacteria translocation in trauma and gut microbiota's effect on inflammation in injury, especially in nervous system injury. Discussion: The impact of gut microbiota on molecular and pathological mechanism of inflammation is the focus now. In addition, the experiments of novel therapies based on gut microbiota's impact on trauma are being carried out. We hope that this study can offer a birds-eye view of this field and promote the gradual improvement of it.

Neural Reflectance Capture in the View-Illumination Domain.

We propose a novel framework to efficiently capture the unknown reflectance on a non-planar 3D object, by learning to probe the 4D view-lighting domain with a high-performance illumination multiplexing setup. The core of our framework is a deep neural network, specifically tailored to exploit the multi-view coherence for efficiency. It takes as input the photometric measurements of a surface point under learned lighting patterns at different views, automatically aggregates the information and reconstructs the anisotropic reflectance. We also evaluate the impact of different sampling parameters over our network. The effectiveness of our framework is demonstrated on high-quality reconstructions of a variety of physical objects, with an acquisition efficiency outperforming state-of-the-art techniques.

High-loading Gα13-binding EXE peptide nanoparticles prevent thrombosis and protect mice from cardiac ischemia/reperfusion injury.

Inefficient delivery is a major obstacle to the development of peptide-based drugs targeting the intracellular compartment. We recently showed that selectively inhibiting integrin outside-in signaling using a peptide (mP6) derived from the Gα13-binding ExE motif within the integrin ß3 cytoplasmic domain had antithrombotic effects. Here, we engineered lipid-stabilized, high-loading peptide nanoparticles (HLPN), in which a redesigned ExE peptide (M3mP6) constituted up to 70% of the total nanoparticle molarity, allowing efficient in vivo delivery. We observed that M3mP6 HLPN inhibited occlusive thrombosis more potently than a clopidogrel/aspirin combination without adverse effects on hemostasis in rodents. Furthermore, M3mP6 HLPN synergized with P2Y12 receptor inhibitors or the clopidogrel/aspirin combination in preventing thrombosis, without exacerbating hemorrhage. M3mP6 HLPN also inhibited intravascular coagulation more potently than the P2Y12 inhibitor cangrelor. Postischemia injection of M3mP6 HLPN protected the heart from myocardial ischemia-reperfusion injury in a mouse model. This study demonstrates an efficient in vivo peptide delivery strategy for a therapeutic that not only efficaciously prevented thrombosis with minimal bleeding risk but also protected from myocardial ischemia-reperfusion injury in mice.

In Situ Growth of Ultrafine Pt Nanoparticles onto Hierarchical Co3 O4 Nanosheet-Assembled Microflowers for Efficient Electrocatalytic Hydrogen Evolution.

The development of Pt-based electrocatalysts with high Pt utilization efficiency toward the hydrogen evolution reaction (HER) is of great significance for the future sustainable hydrogen economy. For rational design of high-performance HER electrocatalyst, the simultaneous consideration of both thermodynamic and kinetic aspects remains greatly challenging. Herein, a simple template-derived strategy is demonstrated for the in situ growth of ultrafine Pt nanoparticles onto Co3 O4 nanosheet-assembled microflowers (abbreviated as Pt/Co3 O4 microflowers hereafter) by using the pre-fabricated PtCo-based Hofmann coordination polymer as reactive templates. The elaborate preparation of such intriguing hierarchical architecture with well-dispersed tiny Pt nanoparticles, abundant metal/oxide heterointerfaces and open configuration endows the formed Pt/Co3 O4 microflowers with high Pt utilization efficiency, rich active sites, lowered energy barrier for water dissociation and expedited reaction kinetics. Consequently, the Pt/Co3 O4 microflowers exhibit superior HER activity with a relatively low overpotential of 34 mV to deliver a current density of 10 mA cm-2 , small Tafel slope (34 mV dec-1 ) and outstanding electrochemical stability, representing an attractive electrocatalyst for practical water splitting. What's more, our concept of in situ construction of metal/oxide heterointerfaces may provide a new opportunity to design high-performance electrocatalysts for a variety of applications.

Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation.

It is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in ß3-/- platelets or by integrin antagonists. The impaired MV release and PS exposure in ß3-/- platelets were rescued by expression of wild-type ß3 but not a Gα13 binding-deficient ß3 mutant (E733EE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα13 or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα13-integrin interaction, suggesting that Gα13-dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα13 delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα13 diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, ß3 integrins serve as a shear sensor activating the Gα13-dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα13-integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation.

Mycobacterium tuberculosis IMPDH in Complexes with Substrates, Products and Antitubercular Compounds.

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5'-monophosphate into xanthosine 5'-monophosphate with the concomitant reduction of NAD+ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD+ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.

A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity.

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization.

Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis.

Microcin C (McC) is heptapeptide adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC, and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its adenosine monophosphate complex reveals specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance.

High-throughput protein purification and quality assessment for crystallization.

The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. "Structural biology-grade" proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are discussed in this chapter.

Cleavable C-terminal His-tag vectors for structure determination.

High-throughput structural genomics projects seek to delineate protein structure space by determining the structure of representatives of all major protein families. Generally this is accomplished by processing numerous proteins through standardized protocols, for the most part involving purification of N-terminally His-tagged proteins. Often proteins that fail this approach are abandoned, but in many cases further effort is warranted because of a protein's intrinsic value. In addition, failure often occurs relatively far into the path to structure determination, and many failed proteins passed the first critical step, expression as a soluble protein. Salvage pathways seek to recoup the investment in this subset of failed proteins through alternative cloning, nested truncations, chemical modification, mutagenesis, screening buffers, ligands and modifying processing steps. To this end we have developed a series of ligation-independent cloning expression vectors that append various cleavable C-terminal tags instead of the conventional N-terminal tags. In an initial set of 16 proteins that failed with an N-terminal appendage, structures were obtained for C-terminally tagged derivatives of five proteins, including an example for which several alternative salvaging steps had failed. The new vectors allow appending C-terminal His(6)-tag and His(6)- and MBP-tags, and are cleavable with TEV or with both TEV and TVMV proteases.

Extracytoplasmic PAS-like domains are common in signal transduction proteins.

We present the crystal structure of the extracytoplasmic domain of the Bacillus subtilis PhoR sensor histidine kinase, part of a two-component system involved in adaptation to low environmental phosphate concentrations. In addition to the PhoR structure, we predict that the majority of the extracytoplasmic domains of B. subtilis sensor kinases will adopt a fold similar to the ubiquitous PAS domain.

Structures of open (R) and close (T) states of prephenate dehydratase (PDT)--implication of allosteric regulation by L-phenylalanine.

The enzyme prephenate dehydratase (PDT) converts prephenate to phenylpyruvate in L-phenylalanine biosynthesis. PDT is allosterically regulated by L-Phe and other amino acids. We report the first crystal structures of PDT from Staphylococcus aureus in a relaxed (R) state and PDT from Chlorobium tepidum in a tense (T) state. The two enzymes show low sequence identity (27.3%) but the same prototypic architecture and domain organization. Both enzymes are tetramers (dimer of dimers) in crystal and solution while a PDT dimer can be regarded as a basic catalytic unit. The N-terminal PDT domain consists of two similar subdomains with a cleft in between, which hosts the highly conserved active site. In one PDT dimer two clefts are aligned to form an extended active site across the dimer interface. Similarly at the interface two ACT regulatory domains create two highly conserved pockets. Upon binding of the L-Phe inside the pockets, PDT transits from an open to a closed conformation.

Diversity and diversification of light chains in myeloma: the specter of amyloidogenesis by proxy.

BACKGROUND/AIMS: Primary amyloidosis and the cancer, multiple myeloma, are characterized by the overproduction of free antibody light chains. Approximately 10% of myeloma patients develop amyloidosis; primary amyloidosis may be thought of as the pathological analog of monoclonal gammopathy of undetermined significance. The kidney is a common site of accumulation of amyloid fibrils and is also the target of other light chain pathologies. Understanding the structural origin of these pathologies is complicated by the extreme primary structure heterogeneity of light chains. METHODS: Patterns of light chain germline gene usage in myeloma patients were compared to those found in other immune system disorders: lymphoma, leukemia, systemic lupus erythematosus and rheumatoid arthritis. RESULTS: Significant differences in apparent gene usage are found in the various diseases; several germline gene products have not been documented in myeloma patients to date. CONCLUSION: The plasma cell dyscrasias including myeloma, lymphoma, leukemia, and monoclonal gammopathy of undetermined significance are usually monoclonal diseases; however, the light chains produced are not homogeneous. Thus, the pathological risk for the patient may change during the course of the illness. Mutation rates in light chains observed during clonal diversification parallel mutations occurring in all genes in the malignant cells and could be a clinically useful biomarker.

A stimulatory role for cGMP-dependent protein kinase in platelet activation.

It is currently accepted that cGMP-dependent protein kinase (PKG) inhibits platelet activation. Here, we show that PKG plays an important stimulatory role in platelet activation. Expression of recombinant PKG in a reconstituted cell model enhanced von Willebrand factor (vWF)-induced activation of the platelet integrin alpha(IIb)beta(3). PKG knockout mice showed impaired platelet responses to vWF or low doses of thrombin and prolonged bleeding time. Human platelet aggregation induced by vWF or low-dose thrombin was inhibited by PKG inhibitors but enhanced by cGMP. Furthermore, a cGMP-enhancing agent, sildenafil, promoted vWF- or thrombin-induced platelet aggregation. The cGMP-stimulated platelet responses are biphasic, consisting of an initial transient stimulatory response that promotes platelet aggregation and a subsequent inhibitory response that limits the size of thrombi.

High throughput methods for gene cloning and expression.

We outline a high throughput process for the production of bacterial expression clones using automated liquid handlers. The protocol consists of a series of interlinked methods representing liquid manipulations or incubations on various stations of the automation system. The methods employ the ligation-independent cloning approach that enables the simultaneous production of plasmids for different expression systems. The current cloning protocol spans 3 days with a linear throughput of 400 targets per production run. This automated approach enables the production of large numbers of bacterial expression clones and ultimately purified proteins. Although they were developed for structural genomics, these molecular protocols can also be applied in high throughput strategies such as those used for site-specific mutagenesis or protein interaction studies.

A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site.

To establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. Automated, plate-based methods for cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of proteins now available from sequenced genomes. Ligation-independent cloning (LIC) is well suited to robotic cloning and expression, but few LIC vectors are available commercially. We have developed a new LIC vector, pMCSG7, that incorporates the tobacco etch virus (TEV) protease cleavage site into the leader sequence. This protease is highly specific and functions under a wide range of conditions. The new vector incorporates an N-terminal his-tag followed by the TEV protease recognition site and a SspI restriction site used for LIC. The vector functioned as expected, giving high cloning efficiencies and strong expression of proteins. Purification and cleavage of a target protein showed that the his-tag and the TEV cleavage site function properly. The protein was purified and cleaved under different conditions to simulate both plate-based screening methods and large-scale purifications for crystal production. The vector also includes a pair of adjacent, unique restriction sites that will allow insertion of additional modules between the his-tag and the cleavage site of the leader sequence to generate a family of vectors suitable for high-throughput production of proteins.