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Selecting an appropriate method for expressing S locus F-box-S2 recombinant protein.

ABSTRACT: A single locus (S locus) including at least two linked genes (female and male determinants) genetically controls the gametophytic self-incompatibility (GSI) in apple, which has evolved to avoid self-fertilization. There has been extensive work done on the female determinant of self-incompatibility, which has led to the determination of the tertiary structure of S-RNase. However, the tertiary structure of male determinant (S locus F-box, SLF/SFB) remains unresolved, which could mainly be due to difficulties associated with its expression in the recombinant expression systems. In addressing this, we have evaluated several in vivo (prokaryotic and eukaryotic) and in vitro expression systems for their efficiency in the expression of apple SLF2. The most successful expression of SLF2 (1 mg/ml) was achieved in E. coli using the synthesized gene in a high salt culture and applying heat shock before induction of culture. We therefore present an approach for the efficient expression of S locus F-box recombinant proteins for future functional and structural studies.

SUBMITTER: Ashkani J

PROVIDER: S-EPMC5480281 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Selecting an appropriate method for expressing S locus F-box-S2 recombinant protein.

Ashkani Jahanshah J Rees D J G DJG

Biotechnology reports (Amsterdam, Netherlands) 20170616

A single locus (S locus) including at least two linked genes (female and male determinants) genetically controls the gametophytic self-incompatibility (GSI) in apple, which has evolved to avoid self-fertilization. There has been extensive work done on the female determinant of self-incompatibility, which has led to the determination of the tertiary structure of S-RNase. However, the tertiary structure of male determinant (S locus F-box, SLF/SFB) remains unresolved, which cou ...[more]

PMID: 28664149

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Project description:UnlabelledWe generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.ImportanceAKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

Project description:BackgroundMost pregnant women in the United States (US) are at risk of inadequate intake of key nutrients during pregnancy from foods alone. Current dietary supplement practices reduce risk of inadequacy for only some nutrients and induce excessive intake of other nutrients.ObjectivesOur study aimed to estimate the doses of supplementation needed to help most pregnant women achieve the recommended intake without exceeding upper limits for key prenatal nutrients and to identify US dietary supplements providing these doses.MethodsWe conducted 24-h dietary recalls in 2450 pregnant participants aged 14-50 y from 2007 to 2019. We estimated the usual intake of vitamins A and D, folate, calcium, iron, and ω-3 FAs from foods alone. We calculated the target doses of supplementation needed to shift 90% of participants to consume above the estimated average requirement and keep 90% below the tolerable upper limit. We identified products in the Dietary Supplement Label Database providing these target doses of supplementation.ResultsThe target dose for supplementation was ≥198 mcg retinol activity equivalents of total vitamin A (with ≤2063 mcg preformed retinol); 7-91 mcg vitamin D; 169-720 mcg dietary folate equivalents of folic acid; 383-943 mg calcium; 13-22 mg iron; and ≥59 mg ω-3 FAs. Out of 20,547 dietary supplements (including 421 prenatal products), 69 products (33 prenatal) contained all 6 nutrients; 7 products (2 prenatal) contained target doses for 5 nutrients. Only 1 product (not a prenatal) contained target doses for all 6 nutrients, but it currently costs ∼USD200/mo and requires 7 tablets per daily serving.ConclusionsAlmost no US dietary supplements provide key nutrients in the doses needed for pregnant women. Affordable and convenient products that fill the gap between food-based intake and estimated requirements of pregnancy without inducing excess intake are needed to support pregnant women and their offspring. Am J Clin Nutr 20XX;xx:xx-xx.

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Selecting an appropriate method for expressing S locus F-box-S2 recombinant protein.

Publications

Selecting an appropriate method for expressing <i>S</i> locus F-box-S2 recombinant protein.

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