THE LOW EXPRESSION LEVEL OF POKEWEED ANTIVIRAL PROTEIN (PAP) GENE IN ESCHERICHIA-COLI BY THE INDUCIBLE LAC PROMOTER IS DUE TO INEFFICIENT TRANSCRIPTION AND TRANSLATION AND NOT TO THE TOXICITY OF THE PAP
J. Xu et al., THE LOW EXPRESSION LEVEL OF POKEWEED ANTIVIRAL PROTEIN (PAP) GENE IN ESCHERICHIA-COLI BY THE INDUCIBLE LAC PROMOTER IS DUE TO INEFFICIENT TRANSCRIPTION AND TRANSLATION AND NOT TO THE TOXICITY OF THE PAP, Archives of biochemistry and biophysics, 351(1), 1998, pp. 82-88
Pokeweed (Phytolacca americana) antiviral protein (PAP) is a glycosida
se which inactivates both eukaryotic and prokaryotic ribosomes. Due to
this activity the wild-type PAP gene encoding mature protein has not
so far been expressed in Escherichia coil. In spite of the ribosome im
pairing activity of the pre-PAP (containing two signal peptides at bot
h termini) on bacterial ribosomes in vitro, the full-length PAP gene h
as been expressed successfully, although at a low level in E. coil und
er an inducible lac promoter. In this study we show that the full-leng
th PAP gene, but not the PAP gene devoid of the N-terminal signal pept
ide codons, can be expressed constitutively in E. coil cells to produc
e a much higher yield as compared with the inducible expression. The f
ull-length PAP is biologically active and it accumulates as inclusion
bodies in bacterial cytoplasm. RNA analysis together with protein meas
urements show that the PAP gene is poorly transcribed and the PAP mRNA
is poorly translated when a lac operator sequence is placed in front
of the Shine/Dalgarno (SD) sequence. Nucleotide folding analysis of th
e 5' untranslated mRNA revealed that the SD sequence in the presence o
f a lac operator is involved in a stable secondary structure, whereas
it is more relaxed in the mRNA transcribed from the constitutive vecto
r. These results provide evidence that the low expression level of ful
l-length PAP gene is due to inefficient transcription and translation
but not to the toxicity of the expressed PAP. (C) 1998 Academic Press.