title: Large-scale production, purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia pastoris.
abstract: The kidney bean lectin Phaseolus vulgaris phytohemagglutinin E-form (PHA-E) was expressed and secreted by the methylotrophic yeast Pichia pastoris. To optimise yields of PHA-E, transformants of P. pastoris were selected for high-level production of the recombinant protein. A scaleable process for the production and purification of gram quantities of recombinant PHA-E is reported. PHA-E was secreted at approximately 100 mg/L at the 2- and 200-L scale and was purified to 95% homogeneity in a single step using cation-exchange chromatography. The purified recombinant PHA-E consists of four forms with molecular masses between 28.5 and 31.5 kDa, as assessed by MALDI-TOF, whereas its native counterpart has a molecular mass of approximately 30.5 kDa. Endoglycosidase treatment revealed that the range in size of the recombinant protein was attributed to differences in the nature of the N-linked oligosaccharides bound to the protein. The primary amino acid sequence of the recombinant PHA-E was found to be identical to the native protein and to have an agglutination activity similar to that of native PHA-E. The data presented here suggest that, using P. pastoris, gram quantities of a recombinant phytohemagglutinin E-form can be produced and that the recombinant protein is similar to the protein synthesised in plants with respect to structure and biological activity.
authors.affiliation: Quality, Health and Nutrition group, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK.
authors.forename: Philippe
authors.initials: P
authors.lastname: Baumgartner
authors.forename: Romaan J M
authors.initials: RJ
authors.lastname: Raemaekers
authors.forename: Alain
authors.initials: A
authors.lastname: Durieux
authors.forename: Angharad
authors.initials: A
authors.lastname: Gatehouse
authors.forename: Howard
authors.initials: H
authors.lastname: Davies
authors.forename: Mark
authors.initials: M
authors.lastname: Taylor
mesh_terms: D000595:Amino Acid Sequence; D004274:DNA, Recombinant / Q000235:genetics; D005818:Genetic Engineering; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D027805:Phaseolus / Q000737:chemistry / Q000235:genetics; D010835:Phytohemagglutinins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D004274:DNA, Recombinant; D010835:Phytohemagglutinins; D011994:Recombinant Proteins