Recombinant pronapin precursor produced in Pichia pastoris displays structural and immunologic equivalent properties to its mature product isolated from rapeseed.
2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor. The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced. The amino-acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser-Glu-Asn. Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris. The induced protein was secreted to the extracellular medium at a yield of 80 mg.L(-1) of culture and was purified by means of size-exclusion chromatography and reverse phase-HPLC. Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein. As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity. The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed. In conclusion, the recombinant expression of napin precursors in P. pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability.
Palomares
Oscar
O
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, Madrid, Spain.
Monsalve
Rafael I
RI
Rodríguez
Rosalía
R
Villalba
Mayte
M
D055516:2S Albumins, Plant; D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D029741:Brassica rapa / Q000737:chemistry*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018744:DNA, Plant; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D016415:Sequence Alignment; D012882:Skin Tests
D055516:2S Albumins, Plant; D000485:Allergens; D018076:DNA, Complementary; D018744:DNA, Plant; D007074:Immunoglobulin G; D010940:Plant Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D007073:Immunoglobulin E; C050363:napin protein, Brassica napus