PMID- 26411458
OWN - NLM
STAT- MEDLINE
DCOM- 20161024
LR  - 20161230
IS  - 1432-0614 (Electronic)
IS  - 0175-7598 (Linking)
VI  - 100
IP  - 2
DP  - 2016 Jan
TI  - Expression of a codon-optimised recombinant Ara h 2.02 peanut allergen in 
      Escherichia coli.
PG  - 661-71
LID - 10.1007/s00253-015-6953-y [doi]
AB  - Current diagnostic tools for peanut allergy using crude peanut extract showed low 
      predictive value and reduced specificity for detection of peanut 
      allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major 
      peanut allergen Ara h 2, contains three IgE epitope recognition sequence of 
      'DPYSPS' and may be a better reagent for component resolve diagnosis. This 
      research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous 
      expression in Escherichia coli and allergenicity study of this recombinant 
      protein. The codon-optimised gene was generated by PCR using overlapping primers 
      and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 
      kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 
      (DE3) host cells was observed upon induction with 1 mM isopropyl 
      beta-D-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was 
      purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) 
      affinity chromatography and refolded by dialysis in decreasing urea 
      concentration, amounting to a yield of 74 mg/l of expression culture. 
      Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and 
      immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 
      2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was 
      able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised 
      Balb/c mice. In addition, the specific IgE antibodies raised against the 
      recombinant protein were able to recognise the native Ara h 2 protein, 
      demonstrating its allergenicity and potential as a reagent for diagnosis and 
      therapeutic study.
FAU - Lew, Min Han
AU  - Lew MH
AD  - Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI 
      Heights, Cheras, Kuala Lumpur, 56000, Malaysia. kelvinlew86213@yahoo.com.
FAU - Lim, Renee Lay Hong
AU  - Lim RL
AD  - Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI 
      Heights, Cheras, Kuala Lumpur, 56000, Malaysia. reneelim@ucsiuniversity.edu.my.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20150928
PL  - Germany
TA  - Appl Microbiol Biotechnol
JT  - Applied microbiology and biotechnology
JID - 8406612
RN  - 0 (2S Albumins, Plant)
RN  - 0 (Allergens)
RN  - 0 (Antigens, Plant)
RN  - 0 (Ara h 2 allergen, Arachis hypogaea)
RN  - 0 (Codon)
RN  - 0 (Epitopes)
RN  - 0 (Glycoproteins)
RN  - 0 (Immunoglobulin G)
RN  - 0 (Plant Proteins)
RN  - 0 (Recombinant Proteins)
RN  - 37341-29-0 (Immunoglobulin E)
SB  - IM
MH  - 2S Albumins, Plant/biosynthesis/chemistry/*genetics/*immunology
MH  - Allergens/biosynthesis/genetics/*immunology
MH  - Amino Acid Sequence
MH  - Animals
MH  - Antigens, Plant/biosynthesis/chemistry/*genetics/*immunology
MH  - Chromatography, Affinity
MH  - *Codon
MH  - Cross Reactions
MH  - Epitopes/immunology
MH  - Escherichia coli/*genetics/metabolism
MH  - Glycoproteins/biosynthesis/chemistry/*genetics/*immunology
MH  - Immunoglobulin E/blood/immunology
MH  - Immunoglobulin G/blood/immunology
MH  - Mice
MH  - Peanut Hypersensitivity/diagnosis/immunology/therapy
MH  - Plant Proteins
MH  - Protein Folding
MH  - Recombinant Proteins/biosynthesis/chemistry/immunology
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
OTO - NOTNLM
OT  - Ara h 2.02
OT  - Codon optimise
OT  - Escherichia coli
OT  - Peanut allergen
OT  - pET-28a (+) vector
EDAT- 2015/09/29 06:00
MHDA- 2016/10/25 06:00
CRDT- 2015/09/29 06:00
PHST- 2015/06/19 00:00 [received]
PHST- 2015/08/22 00:00 [accepted]
PHST- 2015/08/12 00:00 [revised]
PHST- 2015/09/29 06:00 [entrez]
PHST- 2015/09/29 06:00 [pubmed]
PHST- 2016/10/25 06:00 [medline]
AID - 10.1007/s00253-015-6953-y [pii]
AID - 10.1007/s00253-015-6953-y [doi]
PST - ppublish
SO  - Appl Microbiol Biotechnol. 2016 Jan;100(2):661-71. doi: 
      10.1007/s00253-015-6953-y. Epub 2015 Sep 28.