Pichia pastoris is superior to E. coli for the production of recombinant allergenic non-specific lipid-transfer proteins.
Non-specific lipid-transfer proteins (nsLTP) from food and pollen are clinically important allergens, especially in patients recruited from the Mediterranean area. For the use of recombinant nsLTPs in allergy diagnosis and preclinical allergy studies the preparation of nsLTPs in a properly folded and biologically active form is required. Using hazelnut nsLTP (Cor a 8) as a model allergen, heterologous over-expression in Escherichia coli and Pichia pastoris was compared. Recombinant Cor a 8 derived from E. coli and P. pastoris was purified by IMAC and SEC or ammonium sulphate precipitation followed by IEC and SEC, respectively. The recombinant proteins were characterized with regard to IgE-binding by immunoblotting and ELISA, structure by N-terminal sequencing, CD-spectroscopy and LS and to their biological activity using an in vitro basophil histamine release assay. Purification of hazelnut nsLTP from bacterial lysate under native conditions resulted in a low yield of Cor a 8. In addition, the preparation contained non-IgE-reactive aggregations besides the IgE-reactive monomer. In contrast, the yield of rCor a 8 produced in P. pastoris was approximately 270-fold higher and impurities with oligomers have not been detected. Purified monomeric Cor a 8 from bacteria and yeast showed similar IgE-antibody reactivity and secondary structures, and both were capable of inducing histamine release from basophils. In summary, P. pastoris is superior to E. coli as expression system for the production of large quantities of soluble, properly folded, and biologically active rCor a 8.
Pokoj
Sven
S
Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany.
Lauer
Iris
I
Fötisch
Kay
K
Himly
Martin
M
Mari
Adriano
A
Enrique
Ernesto
E
Miguel-Moncin
Maria Del Mar San
Mdel M
Lidholm
Jonas
J
Vieths
Stefan
S
Scheurer
Stephan
S
D000485:Allergens / Q000096:biosynthesis* / Q000737:chemistry; D052179:Antigens, Plant / Q000737:chemistry / Q000378:metabolism; D001681:Biological Assay; D002352:Carrier Proteins / Q000096:biosynthesis*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006636:Histamine Release; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology; D010843:Pichia / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D000485:Allergens; D052179:Antigens, Plant; D002352:Carrier Proteins; C527352:Cor a 8 allergen, Corylus avellana; D011994:Recombinant Proteins; C030363:lipid transfer protein; D007073:Immunoglobulin E