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 ga lactose binding sites were produced in E. coli and targeted to the per iplasm by fusion to the ompA or ompF signal sequences. The proteins we re then isolated from the periplasm and their sugar binding properties assessed. Previous studies investigating the properties of such prote ins produced in Xenopns laevis oocytes suggested that deleted forms of RTB, when not glycosylated, retain their ability to bind simple sugar s, unlike the full-length unglycosylated proteins. When produced in E. coli however we found that only one, EB733, of a number of deleted fo rms of RTB closely related to those previously produced in Xenopus lae vis oocytes, bound to simple sugars. All of the deletion forms of RTB were found to bind in the asialofetuin binding assay; an assay which h as been previously utilized to measure binding of lectins to the termi nal galactose residues of glycoprotein oligosaccharides. However, in c ontrast 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 ass umed that this mutant forms a stable structure similar to that of the C-terminaI domain in the full-length protein. The structural integrity of EB733 was not only suggested by its sugar binding properties and s olubility but also by its consistently higher level of expression and the absence of any apparent susceptibility to E. coli proteases.