Open Access

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

Hai-Yang Yu
Hai-Yang Yu
, 
Dong-Mei Gao
Dong-Mei Gao
, 
Wei Zhou
Wei Zhou
, 
Bing-Bing Xia
Bing-Bing Xia
, 
Zhi-Yuan He
Zhi-Yuan He
, 
Bo Wu
Bo Wu
, 
Min-Zhi Jiang
Min-Zhi Jiang
, 
Ming-Li Wang
Ming-Li Wang
 and 
Jun Zhao
Jun Zhao
   | Jan 29, 2021
Journal of Veterinary Research's Cover Image
About this article
Previous Article
Next Article
Cite
Published Online: Jan 29, 2021
Page range: 101 - 108
Received: Jun 15, 2020
Accepted: Jan 27, 2021
DOI: https://doi.org/10.2478/jvetres-2021-0011
Keywords
© 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

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
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

Fig. 2

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
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

Fig. 4

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

Fig. 5

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%
eISSN:
2450-8608
Language:
English
Publication timeframe:
4 times per year
Journal Subjects:
Life Sciences, Molecular Biology, Microbiology and Virology, other, Medicine, Veterinary Medicine
Journal RSS Feed