Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner

M, Liu; Wang, Bin; Wang, Fei; Liu, Yang; Gao, Dan; Zhang, Chenyao; Ma, Lixin; Yu, Xiaohui

doi:10.1007/s00253-019-09925-6

Cited by 35 publications

(21 citation statements)

References 37 publications

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“…In this construct, PL Pro was fused to the C terminus of superfolder green fluorescent protein (sfGFP) that is known to stabilize a fused partner. [13] A 6×His tag was added to the N terminus of sfGFP for affinity purification using Ni‐NTA resins. A TEV cleavage site was inserted between sfGFP and PL Pro for the proteolytic removal of PL Pro from sfGFP by TEV protease.…”

Section: Resultsmentioning

confidence: 99%

“…To express PL Pro for experimental characterization of existing deubiquitinase and cysteine protease inhibitors, we constructed a PL Pro vector for expression in E. coli (Figure 1A). In this construct, PL Pro was fused to the C terminus of superfolder green fluorescent protein (sfGFP) that is known to stabilize a fused partner [13] . A 6×His tag was added to the N terminus of sfGFP for affinity purification using Ni‐NTA resins.…”

Section: Resultsmentioning

confidence: 99%

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Drug Repurposing for the SARS‐CoV‐2 Papain‐Like Protease

Cho

Lalonde

et al. 2021

ChemMedChem

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As the pathogen of COVID‐19, SARS‐CoV‐2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide products pp1a and pp1ab in the human cell host to form 15 functionally important, mature nonstructural proteins. One of the two enzymes is papain‐like protease or PL Pro . It possesses deubiquitination and deISGylation activities that suppress host innate immune responses toward SARS‐CoV‐2 infection. To repurpose drugs for PL Pro , we experimentally screened libraries of 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PL Pro . Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit strong inhibition of PL Pro at 200 μM. More comprehensive characterizations revealed seven inhibitors GRL0617, SJB2‐043, TCID, DUB‐IN‐1, DUB‐IN‐3, PR‐619, and S130 with an IC 50 value below 40 μM and four inhibitors GRL0617, SJB2‐043, TCID, and PR‐619 with an IC 50 value below 10 μM. Among four inhibitors with an IC 50 value below 10 μM, SJB2‐043 is the most unique in that it does not fully inhibit PL Pro but has a noteworthy IC 50 value of 0.56 μM. SJB2‐043 likely binds to an allosteric site of PL Pro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID‐19 drug repurposing by targeting PL Pro holds promise, but in‐depth analysis of repurposed drugs is necessary to avoid omitting critical allosteric inhibitors.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Drug Repurposing for the SARS‐CoV‐2 Papain‐Like Protease

Cho

Lalonde

et al. 2021

ChemMedChem

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show abstract

“…In this construct, PLpro was fused to the C terminus of superfolder green fluorescent protein (sfGFP) that is known to stabilize a fused partner. 23 A 6×His tag was fused to N terminus of sfGFP for affinity purification using Ni-NTA resins. Between sfGFP and PLpro was a TEV cleavage site for the proteolytic removal of PLpro from the fusion protein by TEV protease.…”

Section: Resultsmentioning

confidence: 99%

Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

Cho¹,

Li²,

Yang³

et al. 2021

Preprint

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As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide translates ORF1a and ORF1ab in human host cells to form 15 functionally important, mature nonstructural proteins. One of the two enzymes, papain-like protease or PLpro, also possesses deubiquitination and deISGylation activities that suppresses host innate immune responses toward SARS-CoV-2 infection. Therefore, PLpro is a potential COVID-19 drug target. To repurpose drugs for PLpro, we experimentally screened 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PLpro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit potent inhibition of PLpro at 200 uM. More comprehensive characterizations revealed 7 inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC50 value below 60 uM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC50 value below 10 uM. Among four inhibitors with an IC50 value below 10 uM, SJB2-043 is the most unique in that it doesn't fully inhibit PLpro but has an outstanding IC50 value of 0.56 uM. SJB2-043 likely binds to an allosteric site of PLpro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PLpro holds promises but in-depth analysis of repurposed drugs is necessary to avoid omitting allosteric inhibitors.

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“…Some disadvantages of this system include a lack of proper post-translational modifications (PTMs), inclusion body (IB) formation, codon bias, and endotoxin issues. Some techniques such as the addition of fusion tags (Liu M. et al, 2019) to the gene sequence, cofactor supplementation, and co-expression of the protein with molecular or chemical chaperones can avoid IB formation (Gupta S. K. et al, 2019) and improve soluble expression (Malekian et al, 2019). Different tags such as Fh8, SUMO, His, TRX, and MBP at the N-or Cterminal enhance protein solubility and also help in affinity purification (Paraskevopoulou and Falcone, 2018).…”

Section: Escherichia Colimentioning

confidence: 99%

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Tripathi

Shrivastava

2019

Front. Bioeng. Biotechnol.

400

243

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Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.

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Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner

Cited by 35 publications

References 37 publications

Drug Repurposing for the SARS‐CoV‐2 Papain‐Like Protease

Drug Repurposing for the SARS‐CoV‐2 Papain‐Like Protease

Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

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