Journal: Applied Microbiology and Biotechnology

Article Title: Enhancing production and assessing IgE reactivity of dog allergen Can f 6 in Pichia pastoris and Escherichia coli

doi: 10.1007/s00253-025-13465-7

Figure Lengend Snippet: E. coli -produced recombinant Can f 6 variant expression and purification. a, b Schematic illustrations of gene expression constructs. a pET28a( +)-Can f 6 expression vector features the Can f 6 gene tagged at the N-terminus with a 6xHis tag. b pET28a( +)-MBP-TEV-Can f 6 expression vector features Can f 6 fused to the C-terminus of a 6xHis-MBP fusion protein, with a TEV protease cleavage site inserted between the fusion partner and the target protein. SDS-PAGE analysis of expression and purification of c rCan f 6 and d rMBP-Can f 6. Lanes: (1), lysate fraction from non-induced E. coli ; (2), lysate fraction from IPTG-induced E. coli ; (3), soluble fraction before purification with nickel-chelate chromatography; (4), soluble fraction after incubation with Ni–NTA agarose; (5–9), wash fractions; (10–14), elution fractions containing purified rCan f 6 and rMBP-Can f 6 proteins, respectively. M—Pierce™ Unstained Protein MW Marker (Thermo Fisher Scientific, USA). Proteins were separated on SDS-PAGE gels of c 12% and d 14% acrylamide concentrations

Article Snippet: Subsequent cloning of the resultant DNA fragment occurred at the BamH I and Xho I sites within pET28a( +) and pET28a( +)-MBP-TEV vectors, gifts from Zita Balklava & Thomas Wassmer (Addgene plasmid #69,929; http://n2t.net/addgene:69929 ; RRID:Addgene_69929) (Currinn et al. ).

Techniques: Produced, Recombinant, Variant Assay, Expressing, Purification, Gene Expression, Construct, Plasmid Preparation, SDS Page, Chromatography, Incubation, Marker