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A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide

Overview of attention for article published in Journal of Venomous Animals and Toxins including Tropical Diseases, June 2015
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Title
A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide
Published in
Journal of Venomous Animals and Toxins including Tropical Diseases, June 2015
DOI 10.1186/s40409-015-0018-7
Pubmed ID
Authors

Herlinda Clement, Vianey Flores, Elia Diego-Garcia, Ligia Corrales-Garcia, Elba Villegas, Gerardo Corzo

Abstract

The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin. The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps. It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a(+). Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer. Both expression systems pQE40 and pET28a(+) expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitro conditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly. The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.

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Mendeley readers

The data shown below were compiled from readership statistics for 52 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 52 100%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 10 19%
Student > Bachelor 10 19%
Researcher 9 17%
Student > Master 5 10%
Student > Doctoral Student 4 8%
Other 4 8%
Unknown 10 19%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 15 29%
Agricultural and Biological Sciences 9 17%
Engineering 3 6%
Chemistry 3 6%
Medicine and Dentistry 3 6%
Other 6 12%
Unknown 13 25%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 29 June 2015.
All research outputs
#7,859,767
of 12,526,623 outputs
Outputs from Journal of Venomous Animals and Toxins including Tropical Diseases
#169
of 321 outputs
Outputs of similar age
#123,723
of 233,308 outputs
Outputs of similar age from Journal of Venomous Animals and Toxins including Tropical Diseases
#2
of 4 outputs
Altmetric has tracked 12,526,623 research outputs across all sources so far. This one is in the 23rd percentile – i.e., 23% of other outputs scored the same or lower than it.
So far Altmetric has tracked 321 research outputs from this source. They receive a mean Attention Score of 3.3. This one is in the 31st percentile – i.e., 31% of its peers scored the same or lower than it.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 233,308 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 37th percentile – i.e., 37% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 4 others from the same source and published within six weeks on either side of this one. This one has scored higher than 2 of them.