Search results for "recombinant"
showing 10 items of 1150 documents
Recombinant laccase from Pediococcus acidilactici CECT 5930 with ability to degrade tyramine
2017
Biogenic amines degradation by bacterial laccases is little known, so we have cloned and heterologously expressed, in E. coli, a new laccase from Pediococcus acidilactici CECT 5930 (Lpa5930), a lactic acid bacterium commonly found in foods able to degrade tyramine. The recombinant enzyme has been characterized by physical and biochemical assays. Here we report the optimization of expression and purification procedures of this laccase. DNA encoding sequence of laccase from P. acidilactici was amplified by PCR and cloned into the expression plasmid pET28a for induction by isopropyl-β-D-thiogalactoipyranoside. Protein expression was performed in E. coli BL21(DE3) harboring pGro7 plasmid expres…
Two maize Kip-related proteins differentially interact with, inhibit and are phosphorylated by cyclin D–cyclin-dependent kinase complexes
2017
Highlight Maize Kip-related proteins can be differentially phosphorylated by different cyclin D–cyclin-dependent kinase complexes and this influences their performance as cyclin-dependent kinase inhibitors.
Role of Bacillus thuringiensis Cry1A toxins domains in the binding to the ABCC2 receptor from Spodoptera exigua
2018
Abstract Cry proteins from Bacillus thuringiensis (Bt) have been used to control insect pests either as formulated sprays or as in Bt-crops. However, field-evolved resistance to Bt proteins is threatening the long-term use of Bt products. The SeABCC2 locus has been genetically linked to resistance to a Bt bioinsecticide (Xentari™) in Spodoptera exigua (a mutation producing a truncated form of the transporter lacking an ATP binding domain was found in the resistant insects). Here, we investigated the role of SeABCC2 in the mode of action of Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and two Cry1A-1Ca hybrids by expressing the receptor in Sf21 and HEK293T cell lines. Cell toxicity assays showed that Sf2…
A self-inducible heterologous protein expression system in Escherichia coli
2016
AbstractEscherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter’s transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG inducti…
The nucleotide sequence of a recombinant tomato yellow leaf curl virus strain frequently detected in Sicily isolated from tomato plants carrying the …
2017
In July 2016, an aggressive syndrome of tomato yellow leaf curl disease was reported in Sicily in tomato plants carrying the Ty-1 resistance gene. A total of 34 samples were collected and analyzed. Twenty-seven out of the 34 samples analyzed appeared to contain only recombinant molecules. One full sequence was obtained after cloning. Alignments and plot similarity analysis showed that the genome of the recombinant, named TYLCV-IL[IT:Sic23:16], was mostly derived from tomato yellow leaf curl virus (TYLCV), with a small region of 132 nucleotides in the non-coding region between the stem-loop and the start of the V2 ORF replaced by 124 nucleotides derived from a virus of a different species, t…
Boolean computation in plants using post-translational genetic control and a visual output signal
2018
[EN] Due to autotrophic growing capacity and extremely rich secondary metabolism, plants should be preferred targets of synthetic biology. However, developments in plants usually run below those in other taxonomic groups. In this work we engineered genetic circuits capable of logic YES, OR and AND Boolean computation in plant tissues with a visual output signal. The circuits, which are deployed by means of Agrobacterium tumefaciens, perform with the conditional activity of the MYB transcription factor Roseal from Antirrhinum majus inducing the accumulation of anthocyanins, plant endogenous pigments that are directly visible to the naked eye or accurately quantifiable by spectrophotometric a…
Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNα2 signal peptide
2016
Efficient production and secretion of recombinant proteins in mammalian cell lines relies in a combination of genetic, metabolic and culture strategy factors. The present work assesses the influence of two key genetic components of expression vectors (promoter and signal peptide) on protein production and secretion effciency in HEK293 cells expressing eGFP as a reporter protein. Firstly, the strength of 3 different promoters was evaluated using transient expression methods. Flow cytometry analysis revealed that the highest level of intracellular protein expression was found when eGFP was under the control of CMV promoter, being 3-times higher in comparison to the rest of the promoters teste…
Activation of the plant plasma membrane H+ -ATPase. Is there a direct interaction between lysophosphatidylcholine and the C-terminal part of the enzy…
1996
The antagonistic effects of the fungal toxin beticolin-1 and of L-alpha-lysophosphatidylcholine (lysoPC) were investigated on the plasma membrane H+-ATPase of the plant Arabidopsis thaliana (isoform 2) expressed in yeast, using both wild-type enzyme (AHA2) and C-terminal truncated enzyme (aha2delta92). Phosphohydrolytic activities of both enzymes were inhibited by beticolin-1, with very similar 50% inhibitory concentrations, indicating that the toxin action does not involve the C-terminal located autoinhibitory domain of the proton pump. Egg lysoPC, a compound that activates the H+-ATPase by a mechanism involving the C-terminal part of the protein, was found to be able to reverse the inhibi…
Constitutive expression of clathrin hub hinders elicitor-induced clathrin-mediated endocytosis and defense gene expression in plant cells.
2012
International audience; Endocytosis has been recently implicated in the signaling network associated with the recognition of microbes by plants. In a previous study, we showed that the elicitor cryptogein was able to induce clathrin-mediated endocytosis (CME) in tobacco suspension cells. Herein, we investigate further the induced CME by means of a GFP-tagged clathrin light chain and a CME inhibitor, the hub domain of clathrin heavy chain. Hub constitutive expression does affect neither cell growth nor constitutive endocytosis but abolishes cryptogein-induced CME. Such an inhibition has no impact on early events in the cryptogein signaling pathway but reduces the expression of defense-associ…
A Robust and All-Inclusive Pipeline for Shuffling of Adeno-Associated Viruses.
2018
Adeno-associated viruses (AAV) are attractive templates for engineering of synthetic gene delivery vectors. A particularly powerful technology for breeding of novel vectors with improved properties is DNA family shuffling, i.e., generation of chimeric capsids by homology-driven DNA recombination. Here, to make AAV DNA shuffling available to a wider community, we present a robust experimental and bioinformatical pipeline comprising: (i) standardized and partially codon-optimized plasmids carrying 12 different AAV capsid genes; (ii) a scalable protocol including troubleshooting guide for viral library production; and (iii) the freely available software SALANTO for comprehensive analysis of ch…