Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli.
The cDNA encoding an isoform of the cypress major pollen allergen, Cup a1.02, has been cloned and expressed in Escherichia coli as a N-terminal 6x His-tagged protein. To increase recovery, Cup a1.02 was expressed at high levels exploiting the T5 strong promoter and led to accumulate as inclusion bodies. The insoluble purified aggregates were solubilized in 6 M guanidine hydrochloride, immobilized using nickel-chelating affinity chromatography, and successfully refolded by controlled removal of the chaotropic reagent. Enhanced protein refolding was observed by reducing the protein concentration at 0.6-0.8 mg/ml. SDS-PAGE and gel filtration chromatography indicated an apparent molecular mass of approximately 40 kDa and the occurrence of the protein as monomers. The reconstituted fusion protein displayed the same immunological properties of the native Cup a1.02 protein as proven by IgE immunoreactivity. Immunoblotting, ELISA, and histamine release test showed that the tag did not preclude the protein functionality hence validating its correct three-dimensional folding. The protein fold was also assessed by CD spectroscopy and deconvolution of the spectrum allowed to estimate the secondary structure as a prevalence of beta structures (higher than 60%) and a small contribution from alpha helices (less than 12%). The reported procedure has proven to be useful for the production of multi-milligrams of recombinant Cup a1.02 allergen suitable for structural biology studies and for the molecular and functional characterization of the IgE binding sites.
Rea
Giuseppina
G
Institute of Crystallography, CNR, Consiglio Nazionale delle Ricerche, P.O. Box 10, I-00016 Monterotondo Stazione, Rome, Italy. giuseppina.rea@ic.cnr.it
Iacovacci
Patrizia
P
Ferrante
Paola
P
Zelli
Massimo
M
Brunetto
Barbara
B
Lamba
Doriano
D
Boffi
Alberto
A
Pini
Carlo
C
Federico
Rodolfo
R
D000485:Allergens / Q000737:chemistry*; D052179:Antigens, Plant; D001665:Binding Sites; D015153:Blotting, Western; D002845:Chromatography; D003001:Cloning, Molecular; D029781:Cupressus / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D006632:Histamine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000737:chemistry; D008954:Models, Biological; D009532:Nickel / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D011058:Pollen / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D000485:Allergens; D052179:Antigens, Plant; C419643:Cup a 1 protein, Cupressus arizonica; D018076:DNA, Complementary; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D009532:Nickel; D006632:Histamine