Pen b 26 is one of the allergens produced by Penicillium brevicompactum which is one of the most prevalent indoor airborne fungi and a major source of respiratory problems, including asthma. Pen b 26 was cloned and expressed as an N-terminal as well as a C-terminal Hiss tagged fusion protein in Escherichia coli. This allergen was purified by immobilized Ni2+-affinity chromatography. The purified Pen b 26 was characterized by immunological, biochemical and biophysical methods. C-His(6) tagged Pen b 26 produced several fold greater yield than N-His(6) tagged Pen b 26. The affinity of C-His(6) tagged Pen b 26 for the specific antibody was also 2.75 times higher than N-His(6) tagged Pen b 26. Crown Copyright (C) 2009 Published by Elsevier Inc. All rights reserved.