Isolation of the gene and large-scale expression and purification of recombinant Erythrina cristagalli lectin

Patrick R. Stancombe, Frances C.G. Alexander, Roger Ling, Mary A. Matheson, Clifford Shone, John A. Chaddock

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

Abstract

Using polymerase chain reaction, the coding sequence for Erythrina cristagalli lectin (ECL) has been cloned and expressed in Escherichia coli. The amplified DNA sequence of ECL is highly homologous to that previously reported for Erythrina corallodendron lectin (ECorL), confirming the absence of introns in the ECL gene. The polypeptide sequences of ECL and ECorL have been compared and five amino acids have been identified that differentiate the two proteins. Recombinant E. cristagalli lectin (recECL) was expressed in E. coli from a genomic clone encoding the mature E. cristagalli lectin gene. Constitutive expression localised recombinant protein in inclusion bodies, which were solubilised, and recECL, subsequently refolded and purified by lactose affinity chromatography. Significant advantages were observed for purification from inclusion bodies rather than from a clone optimised to express soluble protein. A large-scale purification scheme has been developed that can prepare functional recECL from inclusion bodies with a yield of 870mg/L culture. By the range of characterisation methods employed in this study, it has been demonstrated that recECL is functionally equivalent to native ECL obtained from the E. cristagalli plant. In addition, characterisation of the binding of radiolabelled recECL to cultured dorsal root ganglia demonstrated that recECL binds to a single pool of receptors.

Original languageEnglish
Pages (from-to)283-292
Number of pages10
JournalProtein Expression and Purification
Volume30
Issue number2
DOIs
Publication statusPublished - 1 Aug 2003

Keywords

  • Bacterial expression system
  • Cloning
  • Erythrina cristagalli
  • Erythrina-related lectins
  • Lactose affinity

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