Production and purification of active snowdrop lectin in Escherichia coli.
Recombinant snowdrop lectin was produced in Escherichia coli from a cDNA clone encoding mature Galanthus nivalis agglutinin. After induction with isopropylthio-beta-D-galactoside, inclusion bodies from E. coli were solubilised and the G. nivalis agglutinin purified by metal-affinity chromatography using a carboxy-terminal hexahistidine tag. The protein was refolded on the metal-affinity column prior to elution. After purification, the recombinant G. nivalis agglutinin agglutinated rabbit erythrocytes to a dilution similar to that determined for 'native' lectin purified from snowdrop, and showed similar specific binding to mannose. The toxicity of the recombinant G. nivalis agglutinin towards rice brown planthopper (Nilaparvata lugens) was shown to be similar to that of 'native' G. nivalis agglutinin when incorporated into an artificial diet. The recombinant G. nivalis agglutinin is thus functionally similar to 'native' snowdrop lectin.
Longstaff
M
M
Axis Genetics, Babraham, Cambridge, UK. ml@axisgenetics.co.uk
Powell
K S
KS
Gatehouse
J A
JA
Raemaekers
R
R
Newell
C A
CA
Hamilton
W D
WD
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001667:Binding, Competitive / Q000502:physiology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D027801:Galanthus; D015870:Gene Expression / Q000235:genetics; D006384:Hemagglutination / Q000187:drug effects; D007313:Insecta / Q000187:drug effects; D007814:Larva / Q000187:drug effects; D037102:Lectins / Q000737:chemistry* / Q000494:pharmacology; D008358:Mannose / Q000378:metabolism; D037241:Mannose-Binding Lectins; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000494:pharmacology; D010572:Pest Control, Biological; D037121:Plant Lectins; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D017421:Sequence Analysis
D001426:Bacterial Proteins; D037102:Lectins; D037241:Mannose-Binding Lectins; D009005:Monosaccharides; D037121:Plant Lectins; D011994:Recombinant Proteins; C063117:snowdrop lectin; D008358:Mannose