High level synthesis of biologically active recombinant trichosanthin in Escherichia coli.
Two forms of recombinant trichosanthin (rTCS) were synthesized in high levels in Escherichia coli by putting the TCS cDNA under the control of a T7 RNA polymerase-directed promoter. Purification schemes were developed to isolate the recombinant protein from both soluble and insoluble fractions. Form I rTCS possessed the mature TCS sequence and had similar biological activities as the natural protein. Its IC50 was approximately 0.13 nM in an in vitro rabbit reticulocyte translational system and a dose of around 35 micrograms protein per 25 g body weight was sufficient to induce complete abortion in mice. Form II rTCS had a propeptide of 19 aa at the C-terminus and was five times less active than Form I in inhibiting protein synthesis by a rabbit reticulocyte lysate.
Zhu
R H
RH
Department of Biochemistry, Chinese University of Hong Kong, Shatin, NT.
Ng
T B
TB
Yeung
H W
HW
Shaw
P C
PC
D000019:Abortifacient Agents / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000187:drug effects; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012270:Ribosomes / Q000187:drug effects; D012995:Solubility; D015978:Trichosanthin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D000019:Abortifacient Agents; D011994:Recombinant Proteins; D015978:Trichosanthin