Expression of soluble and fully functional ricin A chain in Escherichia coli is temperature-sensitive.
Linkage of ricin A chain (RA) to a cell surface binding antibody or other ligand can result in a potent cytotoxic agent. We expressed the primary sequence for RA in Escherichia coli to facilitate production and to obtain protein free of naturally occurring contaminants, i.e. ricin B chain. Differences in the level of expression and in the characteristics of the expressed protein were noted when several different host/vector systems were tested. Recombinant RA (rRA) was expressed directly under control of the phage lambda major leftward promoter (PL) and the E. coli trp promoter. It was also expressed fused to E. coli alkaline phosphatase sequences, both in the same reading frame for secretion and out-of-reading frame for expression in a cistron-like arrangement. Expression in the PL promoter system, which is temperature-regulated, was achieved at 37 degrees C as well as at 42 degrees C. The protein expressed at these different temperatures had grossly different properties. Whereas rRA expressed at 37 degrees C was soluble and fully active, that produced at 42 degrees C was aggregated, insoluble, and reduced in activity. Soluble rRA could be converted to the insoluble form by incubation at 42 degrees C in vivo, but not in vitro. Hence, this difference in properties does not simply reflect an inherent thermal instability of the protein. Conditions present in vivo, including the possible association with other proteins, are apparently required for this effect on rRA.
Piatak
M
M
Cetus Corporation, Emeryville, California 94608.
Lane
J A
JA
Laird
W
W
Bjorn
M J
MJ
Wang
A
A
Williams
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D012276:Ricin / Q000235:genetics* / Q000302:isolation & purification; D012995:Solubility; D013816:Thermodynamics; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012276:Ricin