Expression of tropomyosin from Blattella germanica as a recombinant non-fusion protein in Pichia pastoris and comparison of its IgE reactivity with its native counterpart.
Tropomyosins derived from invertebrates are well-known pan allergens. However, the allergenicities of recombinant tropomyosins are variable. Here, we undertook to compare the IgE-binding reactivities of native and recombinant German cockroach tropomyosins. Native tropomyosin was purified by ammonium sulfate fractionation, hydroxyapatite column chromatography, and electroelution, and recombinant tropomyosin was expressed in Pichia pastoris. The allergenicities of the native and recombinant tropomyosins were compared by ELISA inhibition analysis. Native German cockroach tropomyosin showed 18% IgE-binding reactivity to German cockroach sensitized sera. Recombinant tropomyosin was produced without fusion protein and its N-terminus was blocked like that of the native counterpart. The IgE-binding reactivity of the recombinant was found to be comparable to that of native tropomyosin over the concentration range 1-1000 ng/ml by ELISA inhibition testing. Recombinant German cockroach tropomyosin expressed in Pichia pastoris showed better allergenicity than that expressed in Escherichia coli. Other factors in addition to the structural differences of native and recombinant proteins may also influence the IgE reactivities of tropomyosins.
Jeong
Kyoung Yong
KY
Department of Parasitology and Institute of Tropical Medicine, Brain Korea 21 Project for Medical Science, Yonsei University, College of Medicine, Seoul, Republic of Korea. jeongky@hitel.net
Lee
Jongweon
J
Lee
In-Yong
IY
Hong
Chein-Soo
CS
Ree
Han-Il
HI
Yong
Tai-Soon
TS
D000485:Allergens / Q000737:chemistry; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D002845:Chromatography; D003058:Cockroaches; D017886:Durapatite / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014335:Tropomyosin / Q000096:biosynthesis* / Q000737:chemistry
D000485:Allergens; D011506:Proteins; D011994:Recombinant Proteins; D014335:Tropomyosin; D007073:Immunoglobulin E; D017886:Durapatite; D000645:Ammonium Sulfate