Cytotoxicity of a recombinant ricin-A-chain fusion protein containing a proteolytically-cleavable spacer sequence.
Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococcal protein A (PA) have been produced in E. coli. Constructs consisting of N-terminal RTA and C-terminal PA (RTA-PA) or N-terminal PA and C-terminal (PA-RTA) were capable of binding to immunoglobulin G (via PA) and of specifically depurinating 28 S ribosomal RNA (via RTA). However, neither fusion protein was cytotoxic to antigen-bearing target cells in the presence of an appropriate monoclonal antibody presumably because the RTA could not be released from the PA within the cytosol where the ribosomal substrate of RTA is located. The overcome this, a short amino acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a disulfide-linked loop containing a trypsin sensitive cleavage site. Cleavage of this fusion protein with trypsin converted the RTA-DT-PA to the two chain form consisting of RTA linked by a disulfide bond to PA. The cleaved fusion protein was highly toxic to Daudi cells coated with anti-immunoglobulin antibody suggesting that the RTA could be released from the PA by reduction within the cytosol.
O'Hare
M
M
Department of Biological Sciences, University of Warwick, Coventry, UK.
Brown
A N
AN
Hussain
K
K
Gebhardt
A
A
Watson
G
G
Roberts
L M
LM
Vitetta
E S
ES
Thorpe
P E
PE
Lord
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D002678:Chimera; D004167:Diphtheria Toxin / Q000235:genetics* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007147:Immunoglobulin Light Chains; D007145:Immunoglobulin kappa-Chains; D008969:Molecular Sequence Data; D037121:Plant Lectins; D010957:Plasmids; D014176:Protein Biosynthesis / Q000187:drug effects*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D012276:Ricin / Q000494:pharmacology*; D013205:Staphylococcal Protein A / Q000494:pharmacology*; D014357:Trypsin
D004167:Diphtheria Toxin; D007147:Immunoglobulin Light Chains; D007145:Immunoglobulin kappa-Chains; D037121:Plant Lectins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C057975:staphylococcal protein A-ricin A fusion protein, recombinant; D012276:Ricin; D014357:Trypsin