scholarly journals Technical Data of Heterologous Expression and Purification of SARS-CoV-2 Proteases Using Escherichia coli System

Data ◽  
2021 ◽  
Vol 6 (9) ◽  
pp. 99
Author(s):  
Rafida Razali ◽  
Vijay Kumar Subbiah ◽  
Cahyo Budiman
Keyword(s):  
Affinity Chromatography ◽  
Drug Targets ◽  
Single Step ◽  
Host Cells ◽  
Soluble Form ◽  
Expression And Purification ◽  
Technical Data ◽  
E Coli ◽  
Main Protease ◽  
Chromatography Step

The SARS-CoV-2 coronavirus expresses two essential proteases: firstly, the 3Chymotrypsin-like protease (3CLpro) or main protease (Mpro), and secondly, the papain-like protease (PLpro), both of which are considered as viable drug targets for the inhibition of viral replication. In order to perform drug discovery assays for SARS-CoV-2, it is imperative that efficient methods are established for the production and purification of 3CLpro and PLpro of SARS-CoV-2, designated as 3CLpro-CoV2 and PLpro-CoV2, respectively. This article expands the data collected in the attempts to express SARS-CoV-2 proteases under different conditions and purify them under single-step chromatography. Data showed that the use of E. coli BL21(DE3) strain was sufficient to express 3CLpro-CoV2 in a fully soluble form. Nevertheless, the single affinity chromatography step was only applicable for 3CLpro-CoV2 expressed at 18 °C, with a yield and purification fold of 92% and 49, respectively. Meanwhile, PLpro-CoV2 was successfully expressed in a fully soluble form in either BL21(DE3) or BL21-CodonPlus(DE3) strains. In contrast, the single affinity chromatography step was only applicable for PLpro-CoV2 expressed using E. coli BL21-CodonPlus(DE3) at 18 or 37 °C, with a yield and purification fold of 86% (18 °C) or 83.36% (37 °C) and 112 (18 °C) or 71 (37 °C), respectively. The findings provide a guide for optimizing the production of SARS-CoV-2 proteases of E. coli host cells.

scholarly journals Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

2005 ◽  
Vol 71 (9) ◽  
pp. 5038-5043 ◽  
Author(s):  
Quande Wei ◽  
Young Soo Kim ◽  
Jeong Hyun Seo ◽  
Woong Sik Jang ◽  
In Hee Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
High Performance ◽  
Affinity Purification ◽  
Antimicrobial Activities ◽  
Host Cells ◽  
Functional Study ◽  
Fusion Partner ◽  
Expression And Purification ◽  
E Coli

ABSTRACT Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein (∼33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP (∼2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.

scholarly journals Expression and Purification of Soluble Recombinant Gsttagged Bpsl2774 Protein from Burkholderia Pseudomallei K96243

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Aisyah Mohamed Rehan ◽  
Hanisah Ujang ◽  
Siti Marhamah Drahaman ◽  
Nor Azurah Mat Akhir ◽  
Noraslinda Muhamad Bunnori ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Burkholderia Pseudomallei ◽  
Insertion Site ◽  
Plasmid Vector ◽  
Target Protein ◽  
Mass Spectrometry Analysis ◽  
Soluble Form ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Sufficient Concentration

Introduction: Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease endemic in Southeast Asia and northern Australia. Cases have been reported in Pahang, Johor Bahru and Kedah. The disease is difficult to combat as B. pseudomallei has shown resistance to various antibiotics and much is still not understood about its pathogenicity. It is suggested that investigating the bacterium hypothetical proteins may provide potential new targets for the development of antimicrobials. The gene of interest in this study, BPSL2774, encoding BPSL2774 hypothetical protein, is a target gene that was predicted as essential using transposon-directed insertion site sequencing technique (TraDIS). We aimed to express and purify soluble GST-tagged BPSL2774 protein at sufficient concentration for future functional assays. Materials and method: The BPSL2774 gene has previously been amplified from genomic DNA of B. pseudomallei K96243 and cloned into pDEST15 (GST-tag) plasmid vector. In this work, the clone was transformed into E. coli BL21(DE3) expression strain cells for up-scaled protein preparations in 0.5 L and 1 L cultures. The auto-induction method was adopted for protein expression. GST-tag affinity chromatography was performed for protein purification and the fractions obtained were analyzed using SDS-PAGE. Results: The target protein was successfully expressed in soluble form and its highest concentration from a 0.8 mL elution fraction was at 1.38 mg/mL. Mass spectrometry analysis of 60 kDa coomassie-stained gel band cut confirmed the presence of the soluble expressed target protein, co-purified with E. coli chaperonin proteins, possibly due to their interaction with the target protein. Higher purity can be achieved through further purification steps following initial GST-tag affinity chromatography. Conclusion: The purified protein was at an acceptable purity and at sufficient concentration for use as samples in a glycosyltransferase bioluminescence assay in the near future.

scholarly journals Single-step purification of full-length human androgen receptor

2005 ◽  
Vol 3 (1) ◽  
pp. nrs.03001 ◽  
Author(s):  
Dalia Juzumiene ◽  
Ching-yi Chang ◽  
Daju Fan ◽  
Tanya Hartney ◽  
John D. Norris ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Affinity Chromatography ◽  
Spodoptera Frugiperda ◽  
Single Step ◽  
Full Length ◽  
Structural Studies ◽  
Site Specific ◽  
E Coli ◽  
Human Androgen Receptor

The full-length human androgen receptor with an N-terminal biotin acceptor peptide tag was overexpressed in Spodoptera frugiperda cells in the presence of 1 μM dihydrotestosterone. Site-specific biotinylation of BAP was achieved in vivo by co-expression of E. coli biotin holoenzyme synthetase. The androgen receptor was purified by single-step affinity chromatography using Streptavidin Mutein Matrix under native conditions. The resultant protein was active, stable, 95% homogeneous, and we obtained sufficient yield for use in functional and structural studies.

Expression and purification of immunologically reactive DPPD, a recombinantMycobacterium tuberculosisskin test antigen, usingMycobacterium smegmatisandEscherichia colihost cells

2004 ◽  
Vol 50 (2) ◽  
pp. 97-105 ◽  
Author(s):  
Chao Liu ◽  
Eric Flamoe ◽  
Hong-Jing Chen ◽  
Darrick Carter ◽  
Steven G Reed ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Guinea Pigs ◽  
Plasmid Vector ◽  
Host Cells ◽  
Delayed Type Hypersensitivity ◽  
Hypersensitivity Reactions ◽  
Expression And Purification ◽  
Purified Protein Derivative ◽  
E Coli

DPPD is a Mycobacterium tuberculosis recombinant antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both guinea pigs and humans infected with M. tuberculosis. In addition, earlier clinical studies with DPPD suggested that this molecule could improve the specificity of the tuberculin skin test, which is used as an important aid for the diagnosis of tuberculosis. However, these studies could only be performed with DPPD engineered as a fusion molecule with another Mycobacterium spp. protein because no expression of DPPD could be achieved as a single molecule or as a conventional fusion protein in any commercial system. Although recombinant fusion proteins are in general suitable for several biological studies, they are by definition not ideal for studies involving highly purified and defined polypeptide sequences. Here, we report two alternative approaches for the expression of immunologically reactive recombinant genuine DPPD. The first approach used the rapidly growing, nonpathogenic Mycobacterium smegmatis as host cells transformed with the pSMT3 plasmid vector containing the full-length DPPD gene. The second approach used Escherichia coli transformed with the pET-17b plasmid vector containing the DPPD gene engineered in a three-copy fusion manner in tandem with itself. Though at low levels, expression and purification of immunologically reactive DPPD in M. smegmatis could be achieved. More abundant expression and purification of DPPD as a homo-trimer molecule was achieved in E. coli ([Formula: see text]2 mg/L of bacterial broth cultures). Interestingly, expression could only be achieved in host cells transformed with the DPPD gene containing its leader peptide. However, the expressed proteins lacked the leader sequence, which indicates that processing of the M. tuberculosis DPPD gene was accurately achieved and necessary in both M. smegmatis and E. coli. More importantly, the delayed type hypersensitivity reactions elicited by purified molecules in guinea pigs infected with M. tuberculosis were indistinguishable from that elicited by purified protein derivative. Because the DPPD gene is present only in the tuberculosis-complex organisms of the Mycobacterium genus, these highly purified molecules should be helpful in identifying individuals sensitized with tubercle bacilli.Key words: Mycobacterium tuberculosis, Mycobacterium smegmatis skin test, DTH, DPPD.

scholarly journals Cloning, expression, and purification of chaperone AcrH from Aeromonas hydrophilausing Escherichia coli host cells

2021 ◽  
Vol 63 (6) ◽  
pp. 23-27
Author(s):  
Van Sang Nguyen ◽  
◽  
Thi Uyen Nguyen ◽  
Keyword(s):  
Host Cell ◽  
Economic Value ◽  
Host Cells ◽  
Expression And Purification ◽  
Gene Encoding ◽  
Host Cell Membrane ◽  
E Coli ◽  
Target Membrane ◽  
Nonspecific Interactions ◽  
And Function

Aeromonas hydrophila (A. hydrophila) is a gram-negativebacterium, using the type III secretion system (T3SS). In the T3SS, a key structure is a translocon that inserts into the target membrane and forms a channel for bacterial toxins into the host cell. A. hydrophila is pathogenic to different organisms, including humans and aquatic animals (especially domestic animals with high economic value in Vietnam and the world, such as fishes, shrimps, amphibians). The pore completes the channel from bacteria to host, is composed of a major translocator (AopB) and minor translocator (AopD). These translocators are bound by a small chaperone (AcrH) in bacterial cytosol. AcrH chaperone plays an important role in keeping the high stability of translocators and prevents nonspecific interactions of hydrophobic domains before the pore formed in the host cell membrane. Previous studies only analysed the structure of the AcrH in combination with the AopB, but in a non-binding form with the AopB has not been elucidated. That limits the understanding of the formation mechanism of T3SS. Therefore, the authors aimed to clone, express, and purify the AcrH recombinant protein which can be used for the structural study and elucidation of T3SS pore formation. In this study, the authors cloned a fragment of the gene encoding for AcrH chaperone from A. hydrophila and inserted the gene into the pET-28a expression vector. AcrH protein from amino acids 21 to 158 was expressed in E. coli BL21 (DE3) and purified using a nickel bead column with high purity (over 99%). As a result, the obtained AcrH protein can be used for studies of structure and function that contribute to perfecting the pathogenesis of gram-negative bacteria and developing research on the treatment mechanism caused by these bacteria.

scholarly journals Expression and Purification of Soluble form of Human Parathyroid Hormone (rhPTH1-34) by Trx Tag in E. coli

2017 ◽  
Vol 5 (3) ◽  
pp. 26-31 ◽  
Author(s):  
Sanaz Yari ◽  
Farida Behzadian ◽  
Hamideh Rouhani nejad ◽  
Mohammad reza Masoumian ◽  
Mahdi Karimi ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Soluble Form ◽  
Human Parathyroid Hormone ◽  
Expression And Purification ◽  
E Coli

scholarly journals The J-Domain of Heat Shock Protein 40 Can Enhance the Transduction Efficiency of Arginine-Rich Cell-Penetrating Peptides

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Tzu-Yin Lin ◽  
Yu-Hsiu Su ◽  
Kun-Hsiung Lee ◽  
Chin-Kai Chuang
Keyword(s):  
Recombinant Proteins ◽  
Mammalian Cells ◽  
Cellular Membrane ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Host Cells ◽  
Soluble Form ◽  
Transduction Efficiency ◽  
E Coli ◽  
Cell Penetrating

Sense and antisense oligonucleotide pairs encoding cell-penetrating peptides PTD(Tat47–57), DPV3A, E162, pVEC, R11, and TP13 were used to construct two sets of pET22b-CPP-DsRed and pET22b-CPP-J-DsRed vectors for CPP-DsRed and CPP-J-DsRed recombinant proteins expression. PTD-DsRed, DPV3A-DsRed, PTD-J-DsRed, and DPV3A-J-DsRed recombinant proteins were expressed in a soluble form. PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins were able to escape fromE. colihost cells into the culture medium. The membrane-penetrating activity of PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins to mammalian cells was more effective than that of PTD-DsRed and DPV3A-DsRed. The route of the cellular membrane translocation of these recombinant proteins is suggested via macropinocytosis followed by an endosomal escape pathway.

Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 main protease by tafenoquine in vitro

2020 ◽  
Author(s):  
Yeh Chen ◽  
Wen-Hao Yang ◽  
Li-Min Huang ◽  
Yu-Chuan Wang ◽  
Chia-Shin Yang ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
High Throughput Screening ◽  
Global Economy ◽  
Drug Targets ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Host Cells ◽  
Compound Libraries ◽  
Main Protease

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the current pandemic, coronavirus disease 2019 (COVID-19), has taken a huge toll on human lives and the global economy. Therefore, effective treatments against this disease are urgently needed. Here, we established a fluorescence resonance energy transfer (FRET)-based high-throughput screening platform to screen compound libraries to identify drugs targeting the SARS-CoV-2 main protease (Mpro), in particular those which are FDA-approved, to be used immediately to treat patients with COVID-19. Mpro has been shown to be one of the most important drug targets among SARS-related coronaviruses as impairment of Mpro blocks processing of viral polyproteins which halts viral replication in host cells. Our findings indicate that the anti-malarial drug tafenoquine (TFQ) induces significant conformational change in SARS-CoV-2 Mpro and diminishes its protease activity. Specifically, TFQ reduces the α-helical content of Mpro, which converts it into an inactive form. Moreover, TFQ greatly inhibits SARS-CoV-2 infection in cell culture system. Hence, the current study provides a mechanistic insight into the mode of action of TFQ against SARS-CoV-2 Mpro. Moreover, the low clinical toxicity of TFQ and its strong antiviral activity against SARS-CoV-2 should warrant further testing in clinical trials.

Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

1983 ◽  
Vol 49 (01) ◽  
pp. 024-027 ◽  
Author(s):  
David Vetterlein ◽  
Gary J Calton
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Culture Media ◽  
Biological Fluids ◽  
Single Step ◽  
High Yield ◽  
Step Method ◽  
Commercial Preparation ◽  
E Coli ◽  
Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

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