Isolation of the gene and large-scale expression and purification of recombinant Erythrina cristagalli lectin.
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 870 mg/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.
Stancombe
Patrick R
PR
Centre for Applied Microbiology and Research, Porton Down, Salisbury, SP4 0JG Wiltshire, UK. patrick.stancombe@camr.org.uk
Alexander
Frances C G
FC
Ling
Roger
R
Matheson
Mary A
MA
Shone
Clifford C
CC
Chaddock
John A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D046508:Culture Techniques; D004895:Erythrina / Q000235:genetics*; D005727:Ganglia, Spinal / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment
D037121:Plant Lectins; D011994:Recombinant Proteins; C045629:erythrina lectin