Ricin B chain fragments expressed in Escherichia coli are able to bind free galactose in contrast to the full length polypeptide.
Deleted forms of ricin B chain (RTB) containing only one of the two galactose binding sites were produced in E. coli and targeted to the periplasm by fusion to the ompA or ompF signal sequences. The proteins were then isolated from the periplasm and their sugar binding properties assessed. Previous studies investigating the properties of such proteins produced in Xenopus laevis oocytes suggested that deleted forms of RTB, when not glycosylated, retain their ability to bind simple sugars, unlike the full-length unglycosylated proteins. When produced in E. coli however we found that only one, EB733, of a number of deleted forms of RTB closely related to those previously produced in Xenopus laevis oocytes, bound to simple sugars. All of the deletion forms of RTB were found to bind in the asialofetuin binding assay; an assay which has been previously utilized to measure binding of lectins to the terminal galactose residues of glycoprotein oligosaccharides. However, in contrast to glycosylated RTB, binding of the deletion mutants could be competed to only a small degree or not at all with galactose. The only deletion mutant observed to bind to free galactose when produced in E. coli corresponded closely to the complete domain 2 of RTB. It is assumed that this mutant forms a stable structure similar to that of the C-terminal domain in the full-length protein. The structural integrity of EB733 was not only suggested by its sugar binding properties and solubility but also by its consistently higher level of expression and the absence of any apparent susceptibility to E. coli proteases.
Wales
R
R
Department of Biological Sciences, University of Warwick, Coventry, UK.
Gorham
H C
HC
Hussain
K
K
Roberts
L M
LM
Lord
J M
JM
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005690:Galactose / Q000378:metabolism*; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis; D012276:Ricin / Q000096:biosynthesis / Q000235:genetics*
D010455:Peptides; D011994:Recombinant Proteins; D012276:Ricin; D005690:Galactose