title: Characterization of purified recombinant Bet v 1 with authentic N-terminus, cloned in fusion with maltose-binding protein.
abstract: A gene encoding the pollen major allergen Bet v 1 from Betula verrucosa (White Birch) has been cloned and expressed in Escherichia coli as a fusion with maltose-binding protein and a Factor Xa proteolytic cleavage site. A generally applicable cloning strategy based on polymerase chain reaction was designed to position the Factor Xa proteolytic site so that the authentic amino terminus of Bet v 1 was generated after cleavage. Fusion protein was isolated by amylose affinity chromatography and enzymatically cleaved by incubation with Factor Xa. Recombinant Bet v 1 was isolated by gel filtration and gave rise to a single band with apparent molecular weight of 17 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequencing of the first 20 amino acids showed complete agreement with the deduced Bet v 1 DNA sequence. Mass spectrometry showed that recombinant Bet v 1 has a molecular mass of 17,440 +/- 2 Da; 86% of the recombinant Bet v 1 amino acid sequence could be verified by digestion with Lys-C and mass spectrometric peptide mapping. The yield of purified recombinant Bet v 1 was 10 mg per liter E. coli cell culture. Two-dimensional gel electrophoresis of purified recombinant protein gave rise to one major protein spot and one or two minor spots focusing at slightly different pHs. The immunochemical properties of recombinant protein were indistinguishable from those of naturally occurring Bet v 1 when compared using a panel of murine monoclonal antibodies and serum IgE from birch pollen allergic patients. Furthermore, recombinant Bet v 1 elicited T-cell proliferation comparable to that of natural Bet v 1. Thus, the methods used for bacterial expression and protein purification result in relatively high yields of folded recombinant Bet v 1 with correct N-terminal sequence and molecular mass. Furthermore, the B- and T-cell epitope structures of recombinant Bet v 1 closely resemble those of the natural protein from pollen.
authors.affiliation: Research Department, ALK-ABELLO Group, Hørsholm, Denmark. Michaels@inet.uni-c.dk
authors.forename: M D
authors.initials: MD
authors.lastname: Spangfort
authors.forename: H
authors.initials: H
authors.lastname: Ipsen
authors.forename: S H
authors.initials: SH
authors.lastname: Sparholt
authors.forename: S
authors.initials: S
authors.lastname: Aasmul-Olsen
authors.forename: M R
authors.initials: MR
authors.lastname: Larsen
authors.forename: E
authors.initials: E
authors.lastname: Mørtz
authors.forename: P
authors.initials: P
authors.lastname: Roepstorff
authors.forename: J N
authors.initials: JN
authors.lastname: Larsen
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000485:Allergens / Q000096:biosynthesis* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D006801:Humans; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011058:Pollen / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013601:T-Lymphocytes / Q000187:drug effects
chemical_list: D018528:ATP-Binding Cassette Transporters; D000485:Allergens; D052179:Antigens, Plant; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C061312:Bet v 1 allergen, Betula; D008320:Maltose