Search results for "Recombinant DNA"

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.

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…

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…

Resistance of a recombinant Escherichia coli to dehydration.

2009

International audience; Dehydration of microorganisms, rendering them anhydrobiotic, is often an efficient method for the short and long term conservation of different strain-producers. However, some biotechnologically important recombinant bacterial strains are extremely sensitive to conventional treatment. We describe appropriate conditions during dehydration of the recombinant Escherichia coli strain HB 101 (GAPDH) that can result dry cells having a 88% viability on rehydration. The methods entails air-drying after addition of 100 mM trehalose to the cultivation medium or distilled water (for short term incubation).

Recombinant factor VIII: past, present and future of treatment of hemophilia A.

2018

The development of recombinant factor VIII (rFVIII) was initially driven by the necessity to treat hemophilia A (HA) patients with FVIII concentrates without the risk of transmitting infectious agents. Over the last three decades the safety of rFVIII has been further improved by completely removing animal or human proteins from the manufacturing process, so that patients would not be exposed to known or emerging pathogens. Recent efforts have concentrated on improving the expression of rFVIII, reducing its immunogenicity and enhancing its pharmacokinetic (PK) behavior. These new goals have been possible thanks to the develop-ment of biotechnology and a better knowledge of the function and s…

IL10 promoter haplotypes may contribute to altered cytokine expression and systemic inflammation in celiac disease

2018

Celiac disease (CD) is an autoimmune/inflammatory condition triggered by dietary gluten intake in genetically predisposed individuals. Though associations with MHC class II HLA-DQ2 or -DQ8 are the primary and necessary genetic predisposition for CD, >97% of genetically predisposed individuals never develop CD. Cytokines were measured in the serum of CD patients and controls. Possible associations with IL10 promoter variants were investigated. Cytokine expression from PBMCs was monitored in response to gluten exposure, or CD3/TCR complex stimulation in the absence or presence of recombinant IL-10. Serum cytokines varied between patients with CD at the time of diagnosis, after dietary elimina…

Real-Life Study for the Diagnosis of House Dust Mite Allergy - The Value of Recombinant Allergen-Based IgE Serology.

2016

<b><i>Background:</i></b><i>Dermatophagoides pteronyssinus</i> is one of the most important perennial allergen sources worldwide. Molecular diagnostics using the commercially available major allergens (Der p 1 and Der p 2) in combination with Der p 10 do not detect house dust mite (HDM) sensitization in a number of cases when used alone. The objective was to evaluate the IgE reactivity profiles of these patients using an experimental immunoassay biochip. <b><i>Methods: </i></b>Sera of HDM-allergic patients (positive skin prick test, CAP class ≥1 for allergen extract, and positive intranasal provocation) were tested for IgE antibodi…

2019

Abstract Tyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (…