Recombinant ricin B chain fragments containing a single galactose binding site retain lectin activity.
Ricin B chain is an N-glycosylated galactose-specific lectin. Examination of the amino acid sequence of the protein has shown it to be the product of a series of gene duplication events based on an original galactose binding peptide. The X-ray crystallographic structure of the protein reveals that it consists of two globular domains, each composed of three smaller subdomains. In each globular domain only one of the three subdomains has retained its ability to bind galactose. Through DNA manipulation we have created a series of fusions of portions of ricin B chain, each carrying only one galactose binding site, to the ricin signal sequence. Transcripts synthesized in vitro using SP6 RNA polymerase were injected into Xenopus oocytes where the recombinant proteins were produced in a mature form. The products were shown to be N-glycosylated and produced in a soluble stable form. Also, they retained the ability to bind galactose. Preliminary experiments on the reassociation of these ricin B chain fragments with ricin A chain to create a modified holotoxin were also carried out.
Wales
R
R
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
Richardson
P T
PT
Roberts
L M
LM
Lord
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003460:Crystallization; D004355:Drug Stability; D005690:Galactose / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012276:Ricin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014162:Transfection; D014961:X-Ray Diffraction; D014982:Xenopus laevis
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012276:Ricin; D005690:Galactose