1. OVEREXPRESSION OF TRUNCATED ARA H2
OVEREXPRESSION OF TRUNCATED ARA H2.02 PEANUT ALLERGEN TIMMY RICHARDO, KELVIN LEW MIN HAN, RENEE LIM LAY HONG* Department of Biotechnology, Faculty of Applied Sciences, UCSI University No,1 Jalan Menara Gading, UCSI Heights, Cheras Kuala Lumpur Malaysia *Corresponding author:
Forward and reverse primer for truncated Arah2.02 was designed using protein solubility predictor software, this statement is wrong, because you don’t design primer using the predictor software. The font size for ‘hydrophobic region’ is too small. Crop the total lysate from the refolding gel picture. Possible to estimate the yield of refolded protein that you obtained, what is the volume of refolded protein obtained from what volume of initial expression culture? Work backwards, ask Min Han, he shld be able to tell you based on the gel intensity.
INTRODUCTION
RESULTS AND DISCUSSION
Peanut is an allergenic food associated with life threatening food-induced allergic reaction (Chatel et al. 2003).
The prevalence of peanut allergy is increasing in Singapore and Philippines and Asian populations (Lehmann et al. 2003).
The peanut allergen Ara h2.02 is the more potent isomer due to the presence of a third IgE binding epitope (Codreau et al. 2011)
Production of a soluble recombinant Ara h2.02 will aid in the development of a diagnostic kit and an animal model for peanut allergy (Chen et al. 2013).
Previously, moderate expression of an insoluble Ara h2.02 in pET-28(+) E. coli was observed (Lew et al., unpublish result).
Truncated Ara h2.02 (A & B) in pET28 constructed
Overexpression of truncated Ara h2.02 Induced with 1mM IPTG at 37OC
M1 M C
M A B
Duration (Hrs)
Duration (Hrs) M M
(bp)
5000
2500
(kDa)
(bp)
25.0
500
416 bp
326 bp
14.2
500
300
300
100
Truncated A Truncated B
Increasing expression level of truncated rAra h2.02 version A and B with time after IPTG induction, higher expression observed for A.
Truncated Ara h2.02 A and B was cloned into Nhe I and Hind III site of pET-28a vector (1 and 2). Positive clone was further confirmed by PCR Screening (A and B).
Truncated A is insoluble however truncated B appears to be partially soluble
Ni-NTA purification and refolding of
recombinant protein A
OBJECTIVE
M I S
M I S
M E E E E E5
M D R
To produce two truncated versions of recombinant Ara h2.02 in pET-28(+) expression system in order to improve yield and solubility of the recombinant protein, while maintaining its IgE epitopes for allergenic property.
(kDa)
(kDa)
(kDa)
(kDa) 25 25 25 25 15 15
METHODOLOGY 15 15
An intense band at 17.2 kDa (A) was eluted at pH 5.3 (E1 to E5). The denatured (D) protein A was refolded (R) and is higher in amount compared to that obtained for full length Ara h2.02.
A B
Expression at 4hr after IPTG induction; Insoluble fraction (I) and soluble fraction (S).
PCR Amplification Two truncated version of Ara h2.02 genes was designed based on the protein solubility profile.
Cloning of into pET-28(+) E. coli expression system
Small scale expression and solubility testing
Truncated proteins (A and B) was produced in pET28(+)/BL21 (DE3) by 1mM IPTG induction.
Ni-NTA Affinity purification of truncated protein containing 6x His-tag
Testing expression and solubility of truncated A and B
(with 1% Glucose and expression at 25oC and 30oC)
A: oC oC
Duration (Hrs)
Duration (Hrs)
Version A
Solubility Test
Truncated protein A remains insoluble despite expression at low temperature and 1% glucose. However, at 25oC and duration of 18hrs, the expression for protein B was increased and remains partially soluble.
M I S
I S M
(kDa)
(kDa)
Version B
IgE epitopes region
1% Glucose: (-) (+) (-) (+) 25
B: oC oC 25
Duration (Hrs)
Duration (Hrs) 15 15
A B
(25oC, 1%Glucose)
I= insoluble, S=soluble
1% Glucose: (-) (+) (-) (+)
CONCLUSION
Truncated recombinant Ara h2.02 A and B proteins were successfully produced at high level and yield compared to the full length protein. Despite slowing down expression level by lowering temperature and adding 1% glucose, protein A remains insoluble; it was successfully purified under denaturing condition and refolded. Truncated protein B on the other hand was partially soluble. Ongoing study include purification of protein B under native condition and characterisation of the allergenicity of both truncated proteins for future applications.
REFERENCES
Chatel, J.-M. et al. (2003) Isolation and Characterisation of Two Complete Ara h 2 Isoforms cDNA. International Archives of Allergy and Immunology, 131(1), pp.14–18
Chen, X. et al. (2011) Analysis of the effector activity of Ara h 2 and Ara h 6 by selective depletion from a crude peanut extract. Journal of Immunological Methods, 372(1-2), 65–70.
Codreanu, F. et al. (2011) A novel immunoassay using recombinant allergens simplifies peanut allergy diagnosis. International Archives of Allergy and Immunology, 154(3), 216–26.
Lehmann, K. et al. (2003) High Yield Expression of E.Coli Purification and Characterisation of Properly Folded Major Peanut Allergen Ara h2. Protein Purification and Expression, 31, pp