Patent ID: 7667009

Claim:
A method for purifying a major mite allergen from Der f 2 obtained by the genetic recombination technique, which comprises the following purification steps (a) to (f): (a) Washing and recovering Escherichia coli cells producing the major mite allergen Der f 2 from culture using MF membrane, disrupting E. coli cells to release their inclusion bodies, washing the inclusion bodies using MF membrane and recovering the inclusion bodies by centrifugation; (b) Dissolving the inclusion bodies using a reducing agent and a denaturing agent, wherein said reducing agent is selected from the group consisting of cysteine, glutathione, dithiothreitol and 2-mercaptoethanol and said denaturing agent is selected from the group consisting of urea and guanidine hydrochloride, and diluting the dissolved inclusion bodies in 10 to 20-fold amount of redox buffer comprising cysteine and cystine; (c) Ultrafiltering the solution using an ultrafiltration membrane of fractionation M.W. of 6,000 to 10,000 to remove low molecular weight components; (d) Passing the solution through a weak anion exchanger column equilibrated with a buffer of pH 7 to 10 and a salt concentration of 0.03 to 0.1M and collecting the non-adsorbed effluent; (e) Contacting the effluent with a hydrophobic column equilibrated with a 2 to 3M sodium chloride buffer of pH 7 to 8, washing the column, and eluting the major mite allergen Der f 2 by decreasing the sodium chloride concentration of the buffer; and (f) Contacting the eluted major mite allergen Der f 2 with a strong anion exchanger column by equilibrating at pH 8.5 to 9.5 and at 1 to 3M urea and then eluting the adsorbed major mite allergen Der f 2 by increasing the salt concentration and dialyzing the major mite allergen Der f 2 with PBS followed by aseptic filtration.