mRNA secondary structure can greatly affect production of recombinant phospholipase A2 toxins in bacteria

Ivanovski, Gabriela; Gubenšek, Franc; Pungerčar, Jože

doi:10.1016/s0041-0101(01)00250-1

Cited by 10 publications

(7 citation statements)

References 20 publications

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“…In addition, the translation results con®rm that the in vitro expression experiments are a valuable method for investigating the effects of RNA structures on RNA±protein interactions (10,16,20,41).…”

Section: Discussionmentioning

confidence: 93%

RNA stem-loop enhanced expression of previously non-expressible genes

Paulus¹

2004

Nucleic Acids Research

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The key step in bacterial translation is formation of the pre-initiation complex. This requires initial contacts between mRNA, fMet-tRNA and the 30S subunit of the ribosome, steps that limit the initiation of translation. Here we report a method for improving translational initiation, which allows expression of several previously non-expressible genes. This method has potential applications in heterologous protein synthesis and high-throughput expression systems. We introduced a synthetic RNA stem±loop (stem length, 7 bp; DG 0 = ±9.9 kcal/mol)

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Section: Discussionmentioning

confidence: 93%

RNA stem-loop enhanced expression of previously non-expressible genes

Paulus¹

2004

Nucleic Acids Research

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“…RT-PCR, ligation, molecular cloning, plasmid DNA extraction and sequencing reactions were performed as previously described [26]. Total genomic DNA from tissue samples (liver, muscles, kidney or skin) was extracted as described by [27].…”

Section: Methodsmentioning

confidence: 99%

Reappraisal of Vipera aspis Venom Neurotoxicity

et al. 2007

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BackgroundThe variation of venom composition with geography is an important aspect of intraspecific variability in the Vipera genus, although causes of this variability remain unclear. The diversity of snake venom is important both for our understanding of venomous snake evolution and for the preparation of relevant antivenoms to treat envenomations. A geographic intraspecific variation in snake venom composition was recently reported for Vipera aspis aspis venom in France. Since 1992, cases of human envenomation after Vipera aspis aspis bites in south-east France involving unexpected neurological signs were regularly reported. The presence of genes encoding PLA2 neurotoxins in the Vaa snake genome led us to investigate any neurological symptom associated with snake bites in other regions of France and in neighboring countries. In parallel, we used several approaches to characterize the venom PLA2 composition of the snakes captured in the same areas.Methodology/Principal FindingsWe conducted an epidemiological survey of snake bites in various regions of France. In parallel, we carried out the analysis of the genes and the transcripts encoding venom PLA2s. We used SELDI technology to study the diversity of PLA2 in various venom samples. Neurological signs (mainly cranial nerve disturbances) were reported after snake bites in three regions of France: Languedoc-Roussillon, Midi-Pyrénées and Provence-Alpes-Côte d'Azur. Genomes of Vipera aspis snakes from south-east France were shown to contain ammodytoxin isoforms never described in the genome of Vipera aspis from other French regions. Surprisingly, transcripts encoding venom neurotoxic PLA2s were found in snakes of Massif Central region. Accordingly, SELDI analysis of PLA2 venom composition confirmed the existence of population of neurotoxic Vipera aspis snakes in the west part of the Massif Central mountains.Conclusions/SignificanceThe association of epidemiological studies to genetic, biochemical and immunochemical analyses of snake venoms allowed a good evaluation of the potential neurotoxicity of snake bites. A correlation was found between the expression of neurological symptoms in humans and the intensity of the cross-reaction of venoms with anti-ammodytoxin antibodies, which is correlated with the level of neurotoxin (vaspin and/or ammodytoxin) expression in the venom. The origin of the two recently identified neurotoxic snake populations is discussed according to venom PLA2 genome and transcriptome data.

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“…Other approaches for enhancing protein expression include 1) manipulation of the nucleotide sequence to alter inhibitory mRNA secondary structure associated with poor translation initiation,36–42 2) overcoming codon usage problems,43 3) selection of appropriate host strains, 4) use of a lower growth temperature44, 45 and 5) use of autoinduction46 or other high‐density media 47…”

Section: Introductionmentioning

confidence: 99%

Enhanced Expression and Purification of Fungal Galactose Oxidase in Escherichia coli and Use for Analysis of a Saturation Mutagenesis Library

Deacon

McPherson

2011

ChemBioChem

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Galactose oxidase (GO) displays broad primary alcohol substrate specificity and so offers potential for engineering new substrate specificity by directed evolution. Producing variant libraries of sufficient complexity ideally requires expression of functional protein in a host such as Escherichia coli. Wild-type GO is produced by the fungus Fusarium graminiarum and is expressed poorly in E. coli. We introduced silent mutations within codons 2-7 of the mature GO coding sequence to enhance GO translation and have combined these with other expression-enhancing mutations. We selected the best E. coli host strain, autoinduction medium, induction temperature, harvest time and cell lysis procedure to produce active recombinant GO. Although normally secreted by the fungus, we have expressed GO in the cytoplasm of E. coli and have used a C-terminal Streptag II sequences for single-step affinity purification. This resulted in purification of 240 mg of functional GO per litre of shake flask culture. We have generated a saturation mutagenesis library at residue Cys383, known to influence substrate binding, and have used the optimised expression conditions to purify and characterise the resulting enzymes.

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mRNA secondary structure can greatly affect production of recombinant phospholipase A2 toxins in bacteria

Cited by 10 publications

References 20 publications

RNA stem-loop enhanced expression of previously non-expressible genes

RNA stem-loop enhanced expression of previously non-expressible genes

Reappraisal of Vipera aspis Venom Neurotoxicity

Enhanced Expression and Purification of Fungal Galactose Oxidase in Escherichia coli and Use for Analysis of a Saturation Mutagenesis Library

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