Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida*☆,☆☆,★,★★
Section snippets
Isolation of total RNA from latex
Fresh latex was collected from regularly tapped Malaysian rubber trees (H. brasiliensis, clone RRIM 600). Latex exuding from the tapped trees was collected while it was continuously mixed with an equal volume of RNA extraction buffer (0.1 mol/L tris–hydrochloric acid, 0.3 mol/L lithium chloride, 1 mmol/L EDTA, 10% SDS, pH 9.5) at ambient temperature. This mixture was centrifuged at 100,000g for 30 minutes at 15°C. The rubber plug was removed, the aqueous phase was extracted with
Cloning and sequence analysis of Hev b 3
Degenerate primers designed according to tryptic peptide sequences of Hev b 3 and sequence similarities with Hev b 1 were used to amplify most of the coding region (amino acids 11 to 204) and the complete 3′ end of a Hev b 3 cDNA by reverse transcription-PCR from total latex RNA. The cDNA sequence was completed at the 5′ end using a protocol to amplify cDNA ends by PCR. The complete cDNA of Hev b 3 consists of 922 bp and contains an open reading frame from bases 105 to 719 (Fig 1). The putative
DISCUSSION
Here we report the cloning and sequencing of Hev b 3, an important Hevea latex protein that is a major allergen in SB-associated latex allergy. We have produced Hev b 3 as a recombinant nonfusion protein in E. coli and demonstrate that the molecule possesses IgE reactivity, which is comparable to nHev b 3. The presence of IgE epitopes on rHev b 3 could be proved by IgE immunoblots and immunoblot inhibition studies (Fig. 4, Fig. 5).
The specificity of IgE binding to rHev b 3 was determined by 2
Acknowledgements
We thank Nyu Ping Chew for technical assistance in Hevea RNA isolation.
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2019, Progress in Molecular Biology and Translational ScienceCitation Excerpt :REF and SRPP are the major hydrophobic paralogous latex proteins but have different aggregation properties and structural features. REF is found to rapidly aggregate under physiological conditions.166 The foremost report illustrating the presence of amyloidogenic proteins in the plant kingdom demonstrates that REF forms micrometer-long, unbranched fibrils at pH 7.4.166
Highlights on Hevea brasiliensis (pro)hevein proteins
2016, BiochimieHevea brasiliensis REF (Hev b 1) and SRPP (Hev b 3): An overview on rubber particle proteins
2014, BiochimieCitation Excerpt :Purification of native REF and SRPP should be performed with care as REF can co-purify with other proteins particularly SRPP [73]. HbREF and HbSRPP may be well expressed in Escherichia coli in the inclusion bodies [33,41,74]. With the possibility of purifying recombinant 6-histidine tagged HbREF and HbSRPP [74] (Fig. 4A), different structures were provided using ATR-FTIR for both proteins in solution (Fig. 4B) or in presence of model membranes [20].
Homologous Hevea brasiliensis REF (Hevb1) and SRPP (Hevb3) present different auto-assembling
2014, Biochimica et Biophysica Acta - Proteins and ProteomicsCitation Excerpt :Compared to the controls, REF and SRPP were not glycosylated in hevea latex, but we could observe few thinner bands above SRPP, and non-proteic bands below 10 kDa (near the front line) that were attributed to glycolipids also present in the monomembrane surrounding the rubber particles [37]. Then, it seems that SRPP is not glycosylated (as REF), and no glycosylation site was detected in SRPP sequence [26]. In addition, it was also previously shown that the recombinant and not glycosylated GST-SRPP is functional and could perfectly help in polyisoprene synthesis [10].
Rubber particle proteins, HbREF and HbSRPP, show different interactions with model membranes
2014, Biochimica et Biophysica Acta - Biomembranes
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Supported in part by the Austrian National Bank, grant No. 7113, and the Austrian Science Foundation, grant No. P12838-GEN.
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Reprint requests: Heimo Breiteneder, PhD, Department of General and Experimental Pathology, AKH-EBO-3Q, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
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*The complete cDNA sequence of Hev b 3 is available from the European Molecular Biology Laboratory database under the accession number AJ223388.
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