Structural and IgE binding analyses of recombinant Der p 2 expressed from the hosts Escherichia coli and Pichia pastoris.
BACKGROUND
The house dust mite allergen Der p 2 is one of the most important indoor allergens associated with allergic disease. Recombinant Der (rDer) p 2 with high IgE binding activity can be readily produced in Escherichia coli and Pichia pastoris, but the structure and IgE binding of the different methods of preparation have not been compared.


METHODS
Secondary structure was assessed by circular dichroism (CD). Intrinsic fluorescence and hydrophobic probe (1-anilinonaphthalene 8-sulphonic acid, ANS) were used to study the Der p 2 hydrophobic cavity. IgE binding was assessed by ELISA inhibition.


RESULTS
CD analysis showed the expected secondary structure for both nDer p 2 and refolded Der p 2 prepared from E. coli inclusion bodies but primarily random structure for Der p 2 secreted from P. pastoris. The secreted product, however, had disulphide bonding and could be refolded to a similar structure to natural Der (nDer) p 2 after precipitation with trichloro-acetic or ammonium sulphate. ANS binding and intrinsic Trp92 fluorescence showed that all recombinant proteins were different to nDer p 2 and that the allergen secreted from P. pastoris did not form a hydrophobic cavity. Despite the marked structural changes, all preparations of Der p 2 had similar IgE binding to nDer p 2.


CONCLUSIONS
Despite almost identical IgE binding, rDer p 2 prepared from both E. coli and P. pastoris showed structural differences to nDer p 2. Der p 2 secreted from P. pastoris lacked most of the natural structure, but refolding could induce the natural structure.
Institute of Molecular Biology and Genetics, Mahidol University, Salaya, Thailand.
S
S
Tanyaratsrisakul
N
N
Malainual
O
O
Jirapongsananuruk
W A
WA
Smith
W R
WR
Thomas
S
S
Piboonpocanun
D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry* / Q000276:immunology; D060829:Arthropod Proteins; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D007073:Immunoglobulin E / Q000276:immunology*; D010843:Pichia; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000276:immunology
D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; C451163:Dermatophagoides pteronyssinus antigen p 2; D011994:Recombinant Proteins; D007073:Immunoglobulin E