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.
Pokeweed (Phytolacca americana) antiviral protein (PAP) is a glycosidase 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 coli. In spite of the ribosome impairing activity of the pre-PAP (containing two signal peptides at both termini) on bacterial ribosomes in vitro, the full-length PAP gene has been expressed successfully, although at a low level in E. coli under an inducible lac promoter. In this study we show that the full-length PAP gene, but not the PAP gene devoid of the N-terminal signal peptide codons, can be expressed constitutively in E. coli cells to produce a much higher yield as compared with the inducible expression. The full-length PAP is biologically active and it accumulates as inclusion bodies in bacterial cytoplasm. RNA analysis together with protein measurements 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 the 5' untranslated mRNA revealed that the SD sequence in the presence of a lac operator is involved in a stable secondary structure, whereas it is more relaxed in the mRNA transcribed from the constitutive vector. These results provide evidence that the low expression level of full-length PAP gene is due to inefficient transcription and translation but not to the toxicity of the expressed PAP.
Xu
J
J
Department of Botany, University of Toronto, Ontario, Canada.
Kaloyanova
D
D
Ivanov
I G
IG
AbouHaidar
M G
MG
D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure; D015870:Gene Expression; D005822:Genetic Vectors; D009699:N-Glycosyl Hydrolases; D009690:Nucleic Acid Conformation; D010940:Plant Proteins / Q000235:genetics* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins; D015183:Restriction Mapping; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000187:drug effects; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics*
D010940:Plant Proteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D001616:beta-Galactosidase; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein