Phage display of ricin B chain and its single binding domains: system for screening galactose-binding mutants.
We demonstrate that the B chain of ricin toxin preserves its lectin activity when expressed as a fusion protein on the surface of fd phage. Moreover, B chain, which folds into two topologically similar globular domains, can be dissected into amino-terminal and carboxyl-terminal domains to form single binding domains (SBDs) of B chain, each of which displays specificity for complex galactosides. The specific binding exhibited by the fusion protein of these SBDs was eliminated when amino acid substitutions Gly-46 in SBD1 or Gly-255 in SBD2 for native asparagine were introduced to alter key residues implicated in hydrogen bonding with substrate. These data demonstrate that it is possible to use a prokaryotic expression system to stably express and screen ricin B chain and its SBDs for sugar-binding mutants. Expression of ricin B chain on the surface of fd phage provides a method that can be used to efficiently select mutants with altered binding activities from a randomly generated library.
Swimmer
C
C
ImmunoGen Inc., Cambridge, MA 02139.
Lehar
S M
SM
McCafferty
J
J
Chiswell
D J
DJ
Blättler
W A
WA
Guild
B C
BC
D000595:Amino Acid Sequence; D001212:Asialoglycoproteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003090:Coliphages; D060748:Fetuins; D005690:Galactose / Q000378:metabolism*; D005822:Genetic Vectors; D006031:Glycosylation; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D012276:Ricin / Q000737:chemistry* / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D060748:Fetuins; D037102:Lectins; D011993:Recombinant Fusion Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D012276:Ricin; D005690:Galactose