Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in <i>E. coli</i> and its antiviral effects to H9N2 avian influenza virus

Zhao, Jun; Yu, Haiyang; Zhao, Yu; Li, Fenghua; Zhou, Wei; Xia, Baojia; He, Zhiyuan; Chen, Jason; Jiang, G.-T.; Wang, Ming-Li

doi:10.1080/10826068.2019.1566150

Cited by 6 publications

(3 citation statements)

References 36 publications

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“…Recombinant ChIFNα was also successfully expressed in different E. coli system in previous studies. For example, Zhao et al also constructed pET32a/ChIFNα vector and succeeded in soluble expression in E. coli Rosetta (DE3) system (Zhao et al, 2019). This is not consistent with our results.…”

Section: Rapid Purification Of the Recombinant Chifnα Proteincontrasting

confidence: 84%

“…Interferons (IFNs) are a large family of glycoproteins produced by immune cells and with antiviral, anti-tumor, and immunomodulatory functions (Zhao et al, 2019). Type I IFNs may work by promoting the transcription of IFN-stimulating genes (ISGs), leading to the production of antiviral proteins such as myxovirus resistance protein (Mx) and 2'-5 ' oligoadenylate synthetases (2',5'-OAS) (Katze et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Cloning, expression and rapid purification of recombinant chicken interferon-alpha

Thao¹,

Truong

Chia

et al. 2023

AJB

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The poultry industry in Vietnam is vulnerable to many viral diseases. Since antibiotics and vaccination provide inadequate protection, it will be beneficial to have alternative immunostimulants to boost non-specific immunity in poultry. Chicken interferon-alpha (ChIFNα) has been described previously with antiviral activity against many pathogens. Therefore, the purpose of this study is to clone and express recombinant ChIFNα in Escherichia coli. The ChIFNα gene was successfully cloned into the pET32a(+) vector, then expressed in the E. coli Rosetta strain. Different expression conditions were tested for the best yield of the expressed protein. Results showed that recombinant ChIFNα was expressed in Rosetta E. coli as inclusion bodies with a yield of 30 mg/100 L culture after induction with 0.5 mM IPTG in 4 hours at 37 oC. The recombinant protein was purified using affinity column chromatography under denaturing conditions with the purity > 94%. Western blot analysis indicated that recombinant ChIFNα reacted specifically with its antibody. Further studies will be carried out to characterize the biological activities of recombinant ChIFNα and its application in the poultry industry.

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Section: Rapid Purification Of the Recombinant Chifnα Proteincontrasting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Cloning, expression and rapid purification of recombinant chicken interferon-alpha

Thao¹,

Truong

Chia

et al. 2023

AJB

View full text Add to dashboard Cite

show abstract

“…As shown in Table 2 [ 21 , [160] , [161] , [162] , [163] , [164] , [165] , [166] , [167] , [168] , [169] , [170] , [171] , [172] , [173] , [174] , [175] , [176] , [177] , [178] , [179] , [180] , [181] , [182] , [183] , [184] , [185] , [186] , [187] , [188] , [189] , [190] , [191] , [192] , [193] , [194] , [195] , [196] , [197] , [198] , [199] , [200] , [201] , [202] , [203] , [204] , [205] , [206] , [207] ], there are many Krebs cycle inhibitors with antiviral properties. Here, metformin, according to its well-established and favorable side effect profile and antiviral activity [ [208] , [209] , [210] , [211] , [212] , [213] , [214] , [215] ], seems to be a promising compound.…”

Section: Metabolic Interventions In Covid-19mentioning

confidence: 99%

Can a metabolism-targeted therapeutic intervention successfully subjugate SARS-COV-2? A scientific rational

Mansouri

Rastegari-Pouyani

Ghanbri-Movahed

et al. 2020

Biomedicine & Pharmacotherapy

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A strategy for producing isotopically labeled peptides with antimicrobial activity or with short in vivo lifetime in Escherichia coli

Ren,

Fan,

Han

et al. 2024

Peptide Science

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Engineered Escherichia coli (E. coli) strains have been widely used to produce isotopically labeled peptides for NMR characterization on their structures and interactions. However, production of antimicrobial peptides (AMPs) by E. coli is still challenging, because AMPs are toxic to E. coli host and would lead to cell death after induction. On the other hand, expression of short peptides in E. coli host often encounter problems of the short in vivo lifetime of the peptides, which were rapidly degraded by endogenous enzymes during expression and purification steps. This report presents a practical method for overcoming these bottlenecks to enable E. coli to express AMPs and peptides that have short in vivo lifetime. This design uses the fusion of thioredoxin tags at both the N‐ and C‐termini of the target peptides. The steric effect of the large soluble tags at both ends of the peptide reduces peptide accessibility, thereby enhancing their in vivo stability and eliminating the toxicity associated with AMPs. The approach was validated using an AMP A3K/L7K‐LAH4 (K3K7) and a membrane fusion peptide (FP), which is a segment of the spike protein of SARS‐CoV‐2 and functions in fusing viral membranes and host cell membranes. Fusion expression of K3K7 with a thioredoxin tag only at the N‐terminal resulted in high toxicity to the host cells, leading to impaired cell growth and a failure to obtain expressed fusion protein. In contrast, the fusion proteins from both termini were successfully expressed and purified. In the case of the FP, the fusion of thioredoxin at both termini significantly enhanced its stability, protecting it from enzymatic degradation during expression and purification steps. On the contrary, the FP with thioredoxin fused only at the N‐terminal was found to be unstable in E. coli host strains. As stable isotope labeling on peptide is essentially important in NMR‐based structure and interaction studies, we also demonstrated that the developed approach enables efficient 15N labeling for NMR studies. This strategy may also be extended to produce other challenging peptides.

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Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus

Cited by 6 publications

References 36 publications

Cloning, expression and rapid purification of recombinant chicken interferon-alpha

Cloning, expression and rapid purification of recombinant chicken interferon-alpha

Can a metabolism-targeted therapeutic intervention successfully subjugate SARS-COV-2? A scientific rational

A strategy for producing isotopically labeled peptides with antimicrobial activity or with short in vivo lifetime in Escherichia coli

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