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

Protein Expr Purif. 2002 Dec;26(3):394-405. doi: 10.1016/s1046-5928(02)00555-7.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • DNA, Recombinant / genetics
  • Genetic Engineering
  • Glycosylation
  • Mass Spectrometry
  • Molecular Sequence Data
  • Molecular Weight
  • Phaseolus* / chemistry
  • Phaseolus* / genetics
  • Phytohemagglutinins / biosynthesis
  • Phytohemagglutinins / chemistry
  • Phytohemagglutinins / isolation & purification*
  • Phytohemagglutinins / metabolism*
  • Pichia / genetics*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification*
  • Recombinant Proteins / metabolism*

Substances

  • DNA, Recombinant
  • Phytohemagglutinins
  • Recombinant Proteins