Expression, purification, and bioactivity of a soluble recombinant ovine interferon-tau in Escherichia coli

Construction and identification of the prokaryotic expression plasmid (pET-32a-roIFN-τ) A – Construction of a recombinant prokaryotic expression vector (pET-32a-roIFN-τ) B – RT-PCR amplification profile of the roIFN-τ gene. Lane M – marker (DNA molecular weight standard); lanes 1–4 – RT-PCR product amplified from total ovine mRNA C – Restriction map of the recombinant pET-32a-roIFN-τ expression plasmid. Lane M – marker (DNA molecular weight standard); lane 1 – single XhoI restriction enzyme digestion of the pET-32a-roIFN-τ recombinant plasmid; lanes 2–3 – digestion of the pET-32a-roIFN-τ plasmid using the XhoI and HindIII restriction enzymes

The identification of the recombinant Trx-roIFN-τ fusion protein expression products by SDS-PAGE and Western blot analyses
A – Expression of the recombinant fusion protein. Lane M – protein molecular weight marker; lane 1 – soluble fraction of the bacterial lysate after sonication; lane 2 – insoluble fraction of the bacterial lysate after sonication; lane 3 – total cellular protein lysate from E. coli Rosetta (DE3) without isopropyl-beta-D-thiogalactopyranoside induction
B – Purification of the recombinant fusion protein. Lane M – protein molecular weight marker; lane 1 – negative control; lane 2 – recombinant protein after two-step chromatography purification
C– Identification of the Trx-roIFN-τ recombinant fusion protein. Lane M – molecular weight marker; lane 1 – negative control; lanes 2–5 – purified roIFN-τ after enterokinase digestion at different time points; lane 6 – purified roIFN-τ protein without enterokinase digestion
D – Western blot analysis of the purified protein. Lane M – molecular weight marker; lanes 1–3 – total cellular protein lysate from E. coli Rosetta (DE3) after induction with IPTG at 2 h, 6 h, and 10 h
Blue arrow – dominant 35 kDa band — The identification of the recombinant Trx-roIFN-τ fusion protein expression products by SDS-PAGE and Western blot analyses A – Expression of the recombinant fusion protein. Lane M – protein molecular weight marker; lane 1 – soluble fraction of the bacterial lysate after sonication; lane 2 – insoluble fraction of the bacterial lysate after sonication; lane 3 – total cellular protein lysate from E. coli Rosetta (DE3) without isopropyl-beta-D-thiogalactopyranoside induction B – Purification of the recombinant fusion protein. Lane M – protein molecular weight marker; lane 1 – negative control; lane 2 – recombinant protein after two-step chromatography purification C– Identification of the Trx-roIFN-τ recombinant fusion protein. Lane M – molecular weight marker; lane 1 – negative control; lanes 2–5 – purified roIFN-τ after enterokinase digestion at different time points; lane 6 – purified roIFN-τ protein without enterokinase digestion D – Western blot analysis of the purified protein. Lane M – molecular weight marker; lanes 1–3 – total cellular protein lysate from E. coli Rosetta (DE3) after induction with IPTG at 2 h, 6 h, and 10 h Blue arrow – dominant 35 kDa band

Fig. 3

The purification profiles of the recombinant protein using affinity chromatography and diethylaminoethyl (DEAE) anion exchange chromatography A – Ni2+ affinity chromatography at an absorbance of 280 nm (A280 nm); B – DEAE anion exchange chromatography at an absorbance of 280 nm (A280 nm) The abscissa (X axis) represents time with the unit of minutes (min). The ordinate (Y axis) represents the electrical signal with the unit of mAu (milli-absorbance units). The –UV symbol represents the detected purple ultraviolet curve profile

Purity characterisation of the recombinant fusion protein as determined by high-performance liquid chromatography (HPLC)
Automatic scaling of HPLC chromatography was applied to measure the purity of the purified target fusion protein at a wavelength of 280 nm AU – absorbance unit — Purity characterisation of the recombinant fusion protein as determined by high-performance liquid chromatography (HPLC) Automatic scaling of HPLC chromatography was applied to measure the purity of the purified target fusion protein at a wavelength of 280 nm AU – absorbance unit

Antiviral activity of the recombinant Trx-roIFN-τ fusion protein detected in the MDBK/VSV titration system
A cytopathic effect (CPE) inhibition bioassay with crystal violet staining was performed to detect the antiviral activity of the Trx-roIFN-τ fusion protein, and the results showed 50% CPE inhibition with 1 unit of roIFN-τ and the control groups for the titration of the biological activity of roIFN-τ. Column V – virus control; column C – cell control; columns 1–10 – interferon test sample serially diluted 4-fold from left to right — Antiviral activity of the recombinant Trx-roIFN-τ fusion protein detected in the MDBK/VSV titration system A cytopathic effect (CPE) inhibition bioassay with crystal violet staining was performed to detect the antiviral activity of the Trx-roIFN-τ fusion protein, and the results showed 50% CPE inhibition with 1 unit of roIFN-τ and the control groups for the titration of the biological activity of roIFN-τ. Column V – virus control; column C – cell control; columns 1–10 – interferon test sample serially diluted 4-fold from left to right

Titration results of a recent batch of expressed recombinant ovine interferon-tau

					Grand total
Column	Dilution	Cytopathic effects record (+ or −)*	Number of protections	Number of lesions	Cumulative number of protections	Cumulative number of lesions	Protection ratio	Protection percentage
1	1:4	−,−	8	0	58	0	58/58	100%
2	1:16	−,−	8	0	50	0	50/50	100%
3	1:64	−,−	8	0	42	0	42/42	100%
4	1:256	−,−	8	0	34	0	34/34	100%
5	1:1024	+,−	7	1	26	1	26/27	96.3%
6	1:4096	+,+	6	2	19	3	19/22	86.3%
7	1:16384	+,+	6	2	13	5	13/18	72.2%
8	1:65536	++,+	5	3	7	8	7/15	46.7%
9	1:262144	+++,+++	2	6	2	14	2/16	12.5%
10	1:1048576	++++,++++	0	8	0	22	0/22	0%

Idioma:: Inglés

Calendario de la edición:: 4 veces al año
Temas de la revista:: Ciencias de la vida, Biología molecular, Microbiología y virología, Ciencias de la vida, otros, Medicina, Medicina veterinaria

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Expression, purification, and bioactivity of a soluble recombinant ovine interferon-tau in Escherichia coli

Publicado en línea: 29 ene 2021

Páginas: 101 - 108

Recibido: 15 jun 2020

Aceptado: 27 ene 2021

DOI: https://doi.org/10.2478/jvetres-2021-0011

Palabras clave
ovine interferon-tau, cytopathic effect inhibition assay, soluble expression, purification, antiviral activity

© 2021 H.Y. Yu et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Titration results of a recent batch of expressed recombinant ovine interferon-tau

Expression, purification, and bioactivity of a soluble recombinant ovine interferon-tau in Escherichia coli

Hai-Yang Yu

Dong-Mei Gao

Wei Zhou

Bing-Bing Xia

Zhi-Yuan He

Bo Wu

Min-Zhi Jiang

Ming-Li Wang

Jun Zhao

Publicado en línea: 29 ene 2021

Páginas: 101 - 108

Recibido: 15 jun 2020

Aceptado: 27 ene 2021

DOI: https://doi.org/10.2478/jvetres-2021-0011

Palabras claveovine interferon-tau, cytopathic effect inhibition assay, soluble expression, purification, antiviral activity

© 2021 H.Y. Yu et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Titration results of a recent batch of expressed recombinant ovine interferon-tau

Palabras clave
ovine interferon-tau, cytopathic effect inhibition assay, soluble expression, purification, antiviral activity