Large-scale production, purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia pastoris.
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.
Baumgartner
Philippe
P
Quality, Health and Nutrition group, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK.
Raemaekers
Romaan J M
RJ
Durieux
Alain
A
Gatehouse
Angharad
A
Davies
Howard
H
Taylor
Mark
M
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*
D004274:DNA, Recombinant; D010835:Phytohemagglutinins; D011994:Recombinant Proteins