Enlarged Preparation Of Peanut Allergen Ara H 1 With High Purity

Peanut allergy is a hypersensitivity reaction caused by allergenic proteins of Ara h 1-Ara h 11 in peanut,which are harmful to the health of patients seriously.Among the peanut allergens,Ara h 1 is a major allergen that causes IgE mediated sensitization in up to 90%of patients with peanut allergy.This allergen is a 7S seed storage glycoprotein or vicilin which comprises 12-16%of the total protein content in peanut extracts.Accordingly,it is necessary to purify Ara h 1 with high-purity for the future work.This work was composed of three parts including exploring the purification method of Ara h 1,enlarging the purification at the laboratory-scale,and the pilot-scale design for purification of Ara h 1.The main work,results and conclusions are presented as follows.1.Firstly,the peanut proteins were extracted by precipitation with ammonium sulfate of 0%,80%and 100%,respectively,and three schemes based on chromatography were designed and compared as pre-experiment.The two-step chromatography,in which the protein solution was first applied to the DEAE-Sepharose Fast Flow anion exchange column(1.6cm×36cm,1.5ml/min,500ml,0-300mmol/L NaCl linear salt gradient),followed by appling to hydroxyapatite column(1.6cmX6cm,0.8ml/min,20-100-150-200mmol/L PBS step gradient),was chosen as the laboratory-scale purification method.The two-step chromatography scheme was repeated 3 times,and it was shown that this strategy is very reliable.Additionally,about 120mg of Ara h 1 with a purity up to 90%can be obtained in one purification cycle.Moreover,the MS analysis of Ara h-1 indicated that the 190kD band,64kD band and 33kD band were Ara h 1 trimer,Ara h 1 monomer and Ara h 1 fragment,respectively.This purification approach is high throughput,reliable and easily for handing,and it is suitable to be enlarged for preparing Ara h 1 with high purity.2.The preparation of Ara h 1 was enlarged 10 times by the two-step chromatography,and the coressponding parameters were described as anion exchange chromatography column(3.5cm×36cm),flow rate of 7.2ml/min at 0-300mmol/L NaCl linear salt gradient(2400ml),hydroxyapatite chromatography column(2.6cm X 18cm),flow rate of 1.5ml/min at 20-100-150-200mmol/L PBS step gradient.It was shown that the method has good repeatability and more than one gram of Ara h 1 was obtained in one preparing cycle,and the CD spectroscopy and ANS fluorescence spectroscopy analysis indicated that the Ara h 1 purified from laboratory-scale were no significant structure differences.Moreover,the yeild and purity of Ara h 1 was maintained well.Therefore,the enlarging purification was completed successfully,and the reliable parameters were made for the pilot-scale design.3.A pilot-scale design for the purification of Ara h 1 was carried out based on the laboratory-scale purification parameters.The preparation of Ara h 1 was enlarged 10 times of the laboratory-scale purification,and more than 10g of Ara h 1 was produced in one purification cycle.Equipment selection for the pilot scale design was done,and the process diagram was drawn by AutoCAD software.The pilot-scale design laid a groundwork for the industrial preperation of Ara h 1.