Expression, purification, and immunological characterization of Cr PI.
An efficient preparation of Periplaneta americana nymphae allergen, Cr PI (54 kDa) is described. It was expressed as a GST-tag fusion protein in Escherichia coli, strain BL21 (DE3). Expression of recombinant Cr PI (rCr PI), denaturation/renaturation of the inclusion bodies and the effects of protein and L-arginine concentration on inclusion body aggregation were optimized. The fusion protein was purified by affinity chromatography and size exclusion chromatography, and Cr PI fusion protein was purified to >95%. rCr PI bound strongly to IgE in the sera of individuals with cockroach allergies as shown by western blot and ELISA. Highly refolded and purified recombinant protein was obtained, providing a basis for the large-scale preparation of Cr PI allergen.
College of Life Sciences, ShenZhen University, ShenZhen, 518060, PRC.
Hai-Qiang
HQ
Wu
Zhi-Gang
ZG
Liu
Pi-Xin
PX
Ran
Zhen-Wen
ZW
Zhou
Bo
B
Gao
D000485:Allergens / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D002479:Inclusion Bodies / Q000378:metabolism; D009758:Nymph / Q000276:immunology; D010526:Periplaneta / Q000235:genetics / Q000276:immunology*; D011489:Protein Denaturation; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014020:Tissue Extracts
D000485:Allergens; D011993:Recombinant Fusion Proteins; D014020:Tissue Extracts; D007073:Immunoglobulin E