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Large-scale production, purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia Pastoris

Lookup NU author(s): Romaan Jan Maria Raemaekers, Professor Angharad MR Gatehouse

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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. (C) 2002 Elsevier Science (USA). All rights reserved.


Publication metadata

Author(s): Baumgartner P, Raemaekers RJM, Durieux A, Gatehouse A, Davies H, Taylor M

Publication type: Article

Publication status: Published

Journal: Protein Expression and Purification

Year: 2002

Volume: 26

Issue: 3

Pages: 394-405

ISSN (print): 1046-5928

ISSN (electronic): 1096-0279

Publisher: Academic Press

URL: http://dx.doi.org/10.1016/S1046-5928(02)00555-7

DOI: 10.1016/S1046-5928(02)00555-7


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