The Low Expression Level of Pokeweed Antiviral Protein (PAP) Gene inEscherichia coliby the InduciblelacPromoter Is Due to Inefficient Transcription and Translation and Not to the Toxicity of the PAP

Xu, Jianhua; Kaloyanova, Dora V.; Ivanov, Iván; AbouHaidar, Mounir G.

doi:10.1006/abbi.1997.0552

Cited by 11 publications

(5 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recombinant PAP contained in the solubilized inclusion bodies was successfully renatured by slowly dialyzing at pH 8.0 buffer in the presence of glycerol and then purified by gel filtration. In contrast to the previously reported low yields (0.3 to 0.9 mg/L) in E. coli, yeast, and transgenic plants (23,25,27), our E. coli expression system allows us to routinely generate 10 to 12 mg/L of recombinant PAP and is amenable for large-scale production. The ribosome inhibitory activity of recombinant PAP expressed in E. coli was confirmed using a rabbit reticulocyte lysate in vitro translation system.…”

Section: Discussionmentioning

confidence: 83%

“…Due to this activity, the wild-type PAP gene had not been successfully expressed in E. coli. Previous attempts to express the PAP gene in E. coli and yeast systems have been largely unsuccessful due to very low yield (0.3 to 0.9 mg/L) and PAP's ribosome inhibitory activity against the host (23,25,27). We have cloned the PAP gene, encoding amino acid residues 23-313, into an expression vector, PQE-31 (Qiagen), under a phage T5 promotor and an inducible lac operator and expressed in E. coli MV1190.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

High-Level Expression and Purification of Biologically Active Recombinant Pokeweed Antiviral Protein

Rajamohan

Engstrom

Denton

et al. 1999

Protein Expression and Purification

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

High-Level Expression and Purification of Biologically Active Recombinant Pokeweed Antiviral Protein

Rajamohan

Engstrom

Denton

et al. 1999

Protein Expression and Purification

View full text Add to dashboard Cite

“…Antiviral activity of the recombi nant protein was tested by a local lesion assay using N. glutinosa as test plant and tobacco mosaic virus (TMV) for infection. Protein and virus treatments were done as described by Xu et al [23]. About 50 ng of purified fused product was applied on the test plant leaves with same age, size, and vigor.…”

Section: Methodsmentioning

confidence: 99%

Cloning and Expression of Small cDNA Fragment Encoding Strong Antiviral Peptide from Celosia cristata in Escherichia coli

Gholizadeh

Kohnehrouz

Santha

et al. 2005

Biochemistry (Moscow)

View full text Add to dashboard Cite

A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.

show abstract

“…The lack of an inhibitory effect of PAP I at concentrations of 1 and 4 µg mL -1 is probably due to insufficient biological activity of the protein at these concentrations. Xu et al (1998) measured the antiviral activity of PAP by a local lesion assay using tobacco (Nicotiana glutinosa) and TMV. They adjusted the PAP concentration to be 62.5 ng mL -1 , 125 ng mL -1 , 0.25 µg mL -1 , 0.5 µg mL -1 , 1.0 µg mL -1 , 2 µg mL -1 , and 4 µg mL -1 in 0.1 M phosphate buffer.…”

Section: Figurementioning

confidence: 99%

Pokeweed (Phytolacca americana L.) antiviral protein inhibits Zucchini yellow mosaic virus infection in a dose-dependent manner in squash plants

Sipahioğlu¹,

Kaya²,

Usta³

et al. 2017

Turk J Agric For

View full text Add to dashboard Cite

Pokeweed antiviral protein (PAP) of Phytolacca americana L. (pokeweed) is a single-chain ribosome-inactivating protein (RIP) characterized by its ability to depurinate plant ribosomes. Here, we isolated, cloned, and expressed the ribosome inactivating protein (RIP) gene, designated as pokeweed antiviral protein type 1 (PAP I), from the summer leaves of pokeweed collected from the Black Sea region (Turkey). Our findings presented here provide direct evidence that exogenous application of PAP I causes concentration-dependent inhibition of Zucchini yellow mosaic virus (ZYMV) infection on squash plants. Squash plants were exposed to PAP I protein with and without DMSO for four consecutive days. Regular spraying of approximately 30 kDa recombinant PAP I at 2 µg mL -1 concentration prevented treated plants from mechanical virus infection. PAP I showed antiviral activity in 9 plants out of 15 inoculated plants. Remarkably, simultaneous application of PAP, DMSO, and ZYMV did not prevent virus infection, suggesting that PAP did not have any effect on viral RNA. In the absence of ZYMV the purified peptide was not cytotoxic for squash plants, although a reduction of plant size, possibly caused by host ribosome depurination, was observed.

show abstract

The Low Expression Level of Pokeweed Antiviral Protein (PAP) Gene inEscherichia coliby the InduciblelacPromoter Is Due to Inefficient Transcription and Translation and Not to the Toxicity of the PAP

Cited by 11 publications

References 22 publications

High-Level Expression and Purification of Biologically Active Recombinant Pokeweed Antiviral Protein

High-Level Expression and Purification of Biologically Active Recombinant Pokeweed Antiviral Protein

Cloning and Expression of Small cDNA Fragment Encoding Strong Antiviral Peptide from Celosia cristata in Escherichia coli

Pokeweed (Phytolacca americana L.) antiviral protein inhibits Zucchini yellow mosaic virus infection in a dose-dependent manner in squash plants

Contact Info

Product

Resources

About