RESUMEN
Pigments provide a simple means to rapidly visually ascertain the quantities or presence of specific microbes in a complex community. The selection of pigment-producing colonies that are simple to differentiate from common colony phenotypes provides a high degree of certainty for the identity of pigment-tagged strains. Successful employment of pigment production is dependent on various intrinsic factors related to proper levels of gene expression and pigment production that are not always easy to predict and vary within each microbe. We have constructed a simple transposon system that incorporates the genes for the production of deoxyviolacein, a pigment produced from intracellular reserves of the amino acid tryptophan, to randomly insert these genes throughout the genome. This tool allows the user to select from many thousands of potential sites throughout a bacterial genome for an ideal location to generate the desired amount of pigment. We have applied this system to a small selection of endophytes and other model bacteria to differentiate these strains from complex communities and confirm their presence after several weeks in natural environments. We provide two examples of applications using the pigments to trace strains following introduction into plant tissues or to produce a reporter strain for extracellular nitrogen compound sensing. We recognize that this tool could have far broader utility in other applications and microbes, and describe the methodology for use by the greater scientific community.
Asunto(s)
Elementos Transponibles de ADN , Pigmentos Biológicos , Elementos Transponibles de ADN/genética , Pigmentos Biológicos/metabolismo , Mutagénesis Insercional/métodos , Vectores Genéticos/genética , Bacterias/genética , Bacterias/metabolismo , Bacterias/clasificación , Triptófano/metabolismo , Endófitos/genética , Endófitos/metabolismoRESUMEN
Site-directed insertion is a powerful approach for generating mutant alleles, but low efficiency and the need for customisation for each target has limited its application. To overcome this, we developed a highly efficient targeted insertional mutagenesis system, CRIMP, and an associated plasmid toolkit, CRIMPkit, that disrupts native gene expression by inducing complete transcriptional termination, generating null mutant alleles without inducing genetic compensation. The protocol results in a high frequency of integration events and can generate very early targeted insertions, during the first cell division, producing embryos with expression in one or both halves of the body plan. Fluorescent readout of integration events facilitates selection of successfully mutagenized fish and, subsequently, visual identification of heterozygous and mutant animals. Together, these advances greatly improve the efficacy of generating and studying mutant lines. The CRIMPkit contains 24 ready-to-use plasmid vectors to allow easy and complete mutagenesis of any gene in any reading frame without requiring custom sequences, modification, or subcloning.
Asunto(s)
Sistemas CRISPR-Cas , Mutagénesis Insercional , Plásmidos , Pez Cebra , Mutagénesis Insercional/métodos , Animales , Plásmidos/genética , Pez Cebra/genética , Vectores Genéticos/genética , Edición Génica/métodos , AlelosRESUMEN
The availability of an alternative and efficient genetic editing technology is critical for fundamental research and strain improvement engineering of Streptomyces species, which are prolific producers of complex secondary metabolites with significant pharmaceutical activities. The mobile group II introns are retrotransposons that employ activities of catalytic intron RNAs and intron-encoded reverse transcriptase to precisely insert into DNA target sites through a mechanism known as retrohoming. We here developed a group II intron-based gene editing tool to achieve precise chromosomal gene insertion in Streptomyces. Moreover, by repressing the potential competition of RecA-dependent homologous recombination, we enhanced site-specific insertion efficiency of this tool to 2.38%. Subsequently, we demonstrated the application of this tool by screening and characterizing the secondary metabolite biosynthetic gene cluster (BGC) responsible for synthesizing the red pigment in Streptomyces roseosporus. Accompanied with identifying and inactivating this BGC, we observed that the impair of this cluster promoted cell growth and daptomycin production. Additionally, we applied this tool to activate silent jadomycin BGC in Streptomyces venezuelae. Overall, this work demonstrates the potential of this method as an alternative tool for genetic engineering and cryptic natural product mining in Streptomyces species.
Asunto(s)
Intrones , Familia de Multigenes , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Intrones/genética , Edición Génica/métodos , Mutagénesis Insercional/métodos , Metabolismo Secundario/genética , Vías Biosintéticas/genética , Recombinación HomólogaRESUMEN
RNA-guided DNA writing enzymes offer promise for programmable gene insertion.
Asunto(s)
Proteína 9 Asociada a CRISPR , Edición Génica , Mutagénesis Insercional , ARN Guía de Sistemas CRISPR-Cas , Retroelementos , Sistemas CRISPR-Cas , Edición Génica/métodos , ARN Guía de Sistemas CRISPR-Cas/química , Mutagénesis Insercional/métodos , Proteína 9 Asociada a CRISPR/química , Genoma Humano , HumanosRESUMEN
The cryptic ß-glucoside GFB (bglGFB) operon in Escherichia coli (E. coli) can be activated by mutations arising under starvation conditions in the presence of an aromatic ß-glucoside. This may involve the insertion of an insertion sequence (IS) element into a "stress-induced DNA duplex destabilization" (SIDD) region upstream of the operon promoter, although other types of mutations can also activate the bgl operon. Here, we show that increased expression of the bglG gene, encoding a well-characterized transcriptional antiterminator, dramatically increases the frequency of both IS-mediated and IS-independent Bgl+ mutations occurring on salicin- and arbutin-containing agar plates. Both mutation rates increased with increasing levels of bglG expression but IS-mediated mutations were more prevalent at lower BglG levels. Mutations depended on the presence of both BglG and an aromatic ß-glucoside, and bglG expression did not influence IS insertion in other IS-activated operons tested. The N-terminal mRNA-binding domain of BglG was essential for mutational activation, and alteration of BglG's binding site in the mRNA nearly abolished Bgl+ mutant appearances. Increased bglG expression promoted residual bgl operon expression in parallel with the increases in mutation rates. Possible mechanisms are proposed explaining how BglG enhances the frequencies of bgl operon activating mutations.
Asunto(s)
Arbutina/farmacología , Proteínas Bacterianas/genética , Alcoholes Bencílicos/farmacología , Escherichia coli/crecimiento & desarrollo , Glucósidos/farmacología , Mutagénesis Insercional/métodos , Proteínas de Unión al ARN/genética , Proteínas Bacterianas/química , Técnicas Bacteriológicas , Medios de Cultivo/química , Elementos Transponibles de ADN , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Glucósidos/metabolismo , Operón , Motivos de Unión al ARN , Proteínas de Unión al ARN/químicaRESUMEN
The sugar starvation-inducible rice αAmy3 promoter and signal peptide are widely used to produce valuable recombinant proteins in rice suspension culture cells. Conventionally, the recombinant gene expression cassette is inserted into the genome at random locations by Agrobacterium- or particle bombardment-mediated transformation. CRISPR/Cas9 gene editing enables gene insertion at a precise target site in the genome. In this study the CRISPR/Cas9 approach was modified for intron-targeted insertion by adding an artificial 3' splicing site upstream of the recombinant gene. Knock-in transgenic rice cell lines containing the recombinant GFP gene inserted in intron 1 of αAmy3 were generated. The endogenous αAmy3 promoter regulated recombinant gene expression and the αAmy3 signal peptide directed secretion of the recombinant GFP protein into the culture medium. In addition, the recombinant GFP protein was localized in amyloplasts, identical to the subcellular localization of endogenous αAmy3 reported previously. This modified CRISPR/Cas9 knock-in approach is simple and highly efficient, and the recombinant gene insertion frequency attained 12.5%. The approach can be applied in the production of pharmaceutical proteins in rice suspension cell cultures. The high efficiency of the GFP reporter gene knock-in method and the maintenance of target gene behavior also make the strategy applicable to endogenous gene functional studies in rice.
Asunto(s)
Sistemas CRISPR-Cas , Intrones , Mutagénesis Insercional/métodos , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Productos Agrícolas/genética , Productos Agrícolas/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Plantas Modificadas GenéticamenteRESUMEN
Cancer genome sequencing has identified dozens of mutations with a putative role in lymphomagenesis and leukemogenesis. Validation of driver mutations responsible for B cell neoplasms is complicated by the volume of mutations worthy of investigation and by the complex ways that multiple mutations arising from different stages of B cell development can cooperate. Forward and reverse genetic strategies in mice can provide complementary validation of human driver genes and in some cases comparative genomics of these models with human tumors has directed the identification of new drivers in human malignancies. We review a collection of forward genetic screens performed using insertional mutagenesis, chemical mutagenesis and exome sequencing and discuss how the high coverage of subclonal mutations in insertional mutagenesis screens can identify cooperating mutations at rates not possible using human tumor genomes. We also compare a set of independently conducted screens from Pax5 mutant mice that converge upon a common set of mutations observed in human acute lymphoblastic leukemia (ALL). We also discuss reverse genetic models and screens that use CRISPR-Cas, ORFs and shRNAs to provide high throughput in vivo proof of oncogenic function, with an emphasis on models using adoptive transfer of ex vivo cultured cells. Finally, we summarize mouse models that offer temporal regulation of candidate genes in an in vivo setting to demonstrate the potential of their encoded proteins as therapeutic targets.
Asunto(s)
Leucemia de Células B/genética , Linfoma de Células B/genética , Animales , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Mutagénesis Insercional/métodosRESUMEN
The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Transformación Celular Neoplásica/genética , Queratinocitos/patología , Mutagénesis Insercional/métodos , Análisis de Secuencia de ADN/métodos , Neoplasias Cutáneas/genética , Proteína de Unión a CREB/genética , Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/patología , Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Coactivador 2 del Receptor Nuclear/genética , Neoplasias Cutáneas/patologíaRESUMEN
Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Since autophosphorylation and dephosphorylation of KaiC are central properties for the function of KaiC, we asked whether autophosphorylation is also a property of ArlH proteins. We observed that both ArlH from the euryarchaeon Pyrococcus furiosus (PfArlH) and from the crenarchaeon Sulfolobus acidocaldarius (SaArlH) have autophosphorylation activity. Using a combination of single-molecule fluorescence measurements and biochemical assays, we show that autophosphorylation of ArlH is closely linked to its oligomeric state when bound to hexameric ArlI. These experiments also strongly suggest that ArlH is a hexamer in its ArlI-bound state. Mutagenesis of the putative catalytic residue (Glu-57 in SaArlH) in ArlH results in a reduced autophosphorylation activity and abolished archaellation and motility in S. acidocaldarius, indicating that optimum phosphorylation activity of ArlH is essential for archaellation and motility.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Movimiento , Pyrococcus furiosus/fisiología , Sulfolobus acidocaldarius/fisiología , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Relojes Circadianos , Mutagénesis Insercional/métodos , FosforilaciónRESUMEN
Interbacterial antagonism and communication are driving forces behind microbial community development. In many Gram-negative bacteria, contact-dependent growth inhibition (CDI) systems contribute to these microbial interactions. CDI systems deliver the toxic C-terminus of a large surface exposed protein to the cytoplasm of neighboring bacteria upon cell-contact. Termed the BcpA-CT, import of this toxic effector domain is mediated by specific, yet largely unknown receptors on the recipient cell outer and inner membranes. In this study, we demonstrated that cytoplasmic membrane proteins GltJK, components of a predicted ABC-type transporter, are required for entry of CDI system protein BcpA-2 into Burkholderia multivorans recipient cells. Consistent with current CDI models, gltJK were also required for recipient cell susceptibility to a distinct BcpA-CT that shared sequences within the predicted "translocation domain" of BcpA-2. Strikingly, this translocation domain showed low sequence identity to the analogous region of an Escherichia coli GltJK-utilizing CDI system protein. Our results demonstrated that recipient bacteria expressing E. coli gltJK were resistant to BcpA-2-mediated interbacterial antagonism, suggesting that BcpA-2 specifically recognizes Burkholderia GltJK. Using a series of chimeric proteins, the specificity determinant was mapped to Burkholderia-specific sequences at the GltK C-terminus, providing insight into BcpA transport across the recipient cell cytoplasmic membrane.
Asunto(s)
Proteínas Bacterianas/fisiología , Burkholderia/fisiología , Proteínas de la Membrana/fisiología , Interacciones Microbianas , Adhesión Bacteriana , Fenómenos Fisiológicos Bacterianos , Biopelículas/crecimiento & desarrollo , Burkholderia/patogenicidad , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Mutagénesis Insercional/métodos , Dominios Proteicos , Especificidad de la EspecieRESUMEN
Recently, encouraging data provided long-awaited hope for gene therapy as a cure for sickle cell disease (SCD). Nevertheless, the transient suspension of the bluebird bio gene therapy trial (clinicaltrials.gov: NCT02140554) after participants developed acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) raised concerns. Potential possibilities for these cases include busulfan, insertional mutagenesis, both, or neither. Busulfan was considered the cause in the first reported case because the transgene was not present in the AML/MDS. However, busulfan is unlikely to have contributed to the most recent case. The transgene was present in the patient's malignant cells, indicating they were infused after busulfan treatment. Several lines of evidence suggest an alternative explanation for events in the bluebird bio trial, including that SCD population studies show an increased relative, but a low absolute, risk of AML/MDS. We propose a new hypothesis: after gene therapy for SCD, the stress of switching from homeostatic to regenerative hematopoiesis by transplanted cells drives clonal expansion and leukemogenic transformation of preexisting premalignant clones, eventually resulting in AML/MDS. Evidence validating our hypothesis will support prescreening individuals with SCD for preleukemic progenitors before gene therapy. While presumed viable, safe strategy has been implemented to resume gene therapy in adults with severe SCD, reasonable alternative curative therapy should be considered for children and adults with severe SCD. Currently, open multicenter clinical trials are incorporating nonmyeloablative conditioning, related haploidentical donors, and posttransplantation cyclophosphamide. Preliminary results from these trials appear promising, and National Institutes of Health-sponsored trials are ongoing in individuals with SCD using this platform.
Asunto(s)
Alquilantes/efectos adversos , Anemia de Células Falciformes/terapia , Busulfano/efectos adversos , Terapia Genética/efectos adversos , Leucemia Mieloide Aguda/etiología , Síndromes Mielodisplásicos/etiología , Anemia de Células Falciformes/genética , Ensayos Clínicos como Asunto , Edición Génica/métodos , Terapia Genética/métodos , Humanos , Leucemia Mieloide Aguda/genética , Mutagénesis Insercional/métodos , Síndromes Mielodisplásicos/genéticaRESUMEN
BACKGROUND: Identifying the regulatory factors that control transcriptional activity is a major challenge of gene expression studies. Here, we describe the application of a novel approach for in vivo identification of regulatory proteins that may directly or indirectly control the transcription of a promoter of interest in Streptomyces. RESULTS: A method based on the combination of Tn5 minitransposon-driven random mutagenesis and lux reporter genes was applied for the first time for the Streptomyces genus. As a proof of concept, we studied the topA supercoiling-sensitive promoter, whose activity is dependent on unknown regulatory factors. We found that the sco4804 gene product positively influences topA transcription in S. coelicolor, demonstrating SCO4804 as a novel player in the control of chromosome topology in these bacteria. CONCLUSIONS: Our approach allows the identification of novel Streptomyces regulators that may be critical for the regulation of gene expression in these antibiotic-producing bacteria.
Asunto(s)
Proteínas Bacterianas/genética , Elementos Transponibles de ADN/genética , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Mutagénesis Insercional/métodos , Regiones Promotoras Genéticas , Streptomyces/genética , Prueba de Estudio ConceptualRESUMEN
BACKGROUND: Pseudorabies virus (PRV) is a preferred vector for recombinant vaccine construction. Previously, we generated a TK&gE-deleted PRV (PRVΔTK&gE-AH02) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4 ~ 5 week-old PRV antibody negative piglets and provide rapid and 100 % protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRVTK&gE-AH02 may be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression. RESULTS: In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S(UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 h post infection (P < 0.05). Moreover, at 12 h post infection, cells infected with PRV-S(UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)ΔTK/gE (P = 0.097) and PRV-S(UL46-27)ΔTK/gE (P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRVΔTK&gE-AH02). CONCLUSIONS: This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.
Asunto(s)
Herpesvirus Suido 1/genética , Mutagénesis Insercional/métodos , Virus de la Diarrea Epidémica Porcina/genética , Animales , Células Cultivadas , Cromosomas Artificiales Bacterianos , Expresión Génica , ARN Mensajero/metabolismo , Recombinación Genética , PorcinosRESUMEN
The P. aeruginosa reference strain PAO1 has been used to delineate much of the physiology, metabolism, and fundamental biology of the species. The wild-type parent of PAO1 was lost, and PAO1 carries a regulatory mutation introduced for positive genetic selection that affects antibiotic resistance, virulence, quorum sensing, and other traits. The mutation is a loss-of-function change in an oxidoreductase gene (mexS), which constitutively activates a stress response controlled by a positive regulator (MexT). Fitness defects associated with the constitutive response have led to the inadvertent selection of mexT-minus suppressor mutations, creating genetic heterogeneity in PAO1 sublines studied in different laboratories. To help circumvent complications due to the mexS-minus phenotypes, we created a wild-type version of PAO1 (called LPAO) by "reverting" its mexS to the functional allele likely to have been in its parent. Phenotypic analysis revealed that the mexS-minus allele in PAO1 makes growth sensitive to salt (NaCl) and is lethal when combined with mutations inactivating the major sodium antiporter (ShaABCDEF). The salt sensitivity of PAO1 may underlie some complex mexS-minus phenotypes and help explain the selection of mexT-minus suppressor mutations. To facilitate genetic comparisons of PAO1, LPAO, and other P. aeruginosa strains, we developed a transformation procedure to transfer selectable alleles, such as transposon insertion alleles, between strains. Overall, the study helps explain phenotypic heterogeneity of PAO1-derived strains and provides resources to help recognize and eliminate difficulties due to it. IMPORTANCE The P. aeruginosa reference strain PAO1 carries a regulatory mutation that may affect processes characterized in it. To eliminate complications due to the mutation, we constructed a version of the missing wild-type parent strain and developed methods to transfer mutations between PAO1 and the new strain. The methods are likely to be applicable to other isolates of P. aeruginosa as well.
Asunto(s)
Mutagénesis Insercional/métodos , Pseudomonas aeruginosa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , Regulación Bacteriana de la Expresión Génica , Mutación , Pseudomonas aeruginosa/metabolismo , Transformación BacterianaRESUMEN
Mapping the genomic location to which transposons jumped is of greatest interest to transposon biologists. Transposon display (TD) is the technique of choice that is easy and fast in determining the neo-insertion positions of a target transposon. Essentially, tagging of transposon is performed by digesting genomic DNA, ligating adaptors to digested DNA ends and PCR amplifying genomic regions flanking the transposon of interest. In this chapter, the experimental procedure of TD is described using Onsen retrotransposon of Arabidopsis as an example.
Asunto(s)
Arabidopsis/genética , Mutagénesis Insercional/métodos , Retroelementos , Mapeo Cromosómico , ADN de Plantas/genéticaRESUMEN
Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration occurred randomly at a single locus in the genome and mainly as a single copy. Thus, we confirmed the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci. Furthermore, we often observed variations in the vector sequence itself. From these observations, we speculate that a nonhomologous end-joining-like mechanism is employed during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in this ecologically important marine alga.
Asunto(s)
Chlorophyta/genética , Reparación del ADN/genética , Genoma/genética , Mutación/genética , Reparación del ADN/fisiología , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutagénesis Insercional/métodos , NanoporosRESUMEN
We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This two-step method combines phiC31 integrase-mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.
Asunto(s)
Marcación de Gen/métodos , Mutagénesis Insercional/métodos , Mutagénesis Sitio-Dirigida/métodos , Animales , Sitios de Ligazón Microbiológica/genética , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Drosophila melanogaster , Genoma de los Insectos , Integrasas/genética , Integrasas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Multi-domain proteins are not only formed through natural evolution but can also be generated by recombinant DNA technology. Because many fusion proteins can enhance the selectivity of cell targeting, these artificially produced molecules, called multi-specific biologics, are promising drug candidates, especially for immunotherapy. Moreover, the rational design of domain linkers in fusion proteins is becoming an essential step toward a quantitative understanding of the dynamics in these biopharmaceutics. We developed a computational framework to characterize the impacts of peptide linkers on the dynamics of multi-specific biologics. Specifically, we first constructed a benchmark containing six types of linkers that represent various lengths and degrees of flexibility and used them to connect two natural proteins as a test system. We then projected the microsecond dynamics of these proteins generated from Anton onto a coarse-grained conformational space. We further analyzed the similarity of dynamics among different proteins in this low-dimensional space by a neural-network-based classification model. Finally, we applied hierarchical clustering to place linkers into different subgroups based on the classification results. The clustering results suggest that the length of linkers, which is used to spatially separate different functional modules, plays the most important role in regulating the dynamics of this fusion protein. Given the same number of amino acids, linker flexibility functions as a regulator of protein dynamics. In summary, we illustrated that a new computational strategy can be used to study the dynamics of multi-domain fusion proteins by a combination of long timescale molecular dynamics simulation, coarse-grained feature extraction, and artificial intelligence.
Asunto(s)
Aminoácidos/química , Inteligencia Artificial , Mutagénesis Insercional/métodos , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/química , Aminoácidos/metabolismo , Benchmarking , Biología Computacional/métodos , Expresión Génica , Humanos , Simulación de Dinámica Molecular , Docilidad , Dominios Proteicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
OBJECTIVES: Epithelial growth factor receptor (EGFR), as a malignancy marker, is overly expressed in multiple solid tumors including colorectal neoplasms, one of the most prevalent malignancies worldwide. The main objective of this study is to enhance the efficacy of anti-tumor therapy targeting EGFR by constructing a novel EGFR-specific immunotoxin (C-CUS245C) based on Cetuximab and recombinant Cucurmosin (CUS245C). METHODS: E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS245C with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS245C. The chemical conjugation method was used for the preparation of C-CUS245C. Then dialysis and IMAC were used to purify C-CUS245C. Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS245C. At last the anti-colorectal cancer activity of C-CUS245C was investigated in vitro and in vivo. RESULTS: CUS245C with high purity could be obtained from the prokaryotic system. C-CUS245C was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS245C on EGFR-positive cells for 120 h with IC50 values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS245C was remarkably more potent than that of Cetuximab, CUS245C, and C + CUS245C (P < 0.001). Whereas the cytotoxicity of C-CUS245C could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS245C had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS245C for the targeted therapy of colorectal neoplasms. CONCLUSIONS: C-CUS245C exhibits potent and EGFR-specific cytotoxicity. Insertional mutagenesis technique is worthy to be adopted in the preparation of immunotoxin. Immunotoxin can be highly purified through dialysis followed by IMAC.
Asunto(s)
Cetuximab/uso terapéutico , Neoplasias Colorrectales/terapia , Inmunotoxinas/uso terapéutico , Terapia Molecular Dirigida/métodos , Proteínas de Plantas/uso terapéutico , Animales , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cetuximab/farmacología , Cromatografía de Afinidad/métodos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Escherichia coli/metabolismo , Humanos , Inmunoconjugados/química , Inmunoconjugados/uso terapéutico , Inmunotoxinas/química , Inmunotoxinas/aislamiento & purificación , Inmunotoxinas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutagénesis Insercional/métodos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Genes that play a role in stress response mechanisms and other phenotypes of Listeria monocytogenes can be identified by construction and screening of mutant libraries. In this chapter, we describe the construction and screening of mutant libraries of L. monocytogenes using the plasmid pMC38, carrying a mariner-based transposon system (TC1/mariner) and constructed by Cao et al. (Appl Environ Microbiol 73:2758-2761, 2007). Following screening of mutant libraries, putative mutants are identified and the transposon is localized, leading to identification of the genes responsible for the phenotype of interest. To confirm the role of the transposon-harboring gene in the relevant phenotype, transposon mutants are genetically complemented with the wild-type gene using the site-specific temperature-sensitive integration vector pPL2, constructed by Lauer et al. (J Bacteriol 184:4177-4186, 2002).