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1.
Protein J ; 43(2): 283-297, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38265733

ABSTRACT

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.


Subject(s)
Escherichia coli , Protein Refolding , Recombinant Proteins , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Crystallography, X-Ray , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Crystallization , Agglutinins/chemistry , Agglutinins/genetics , Agglutinins/metabolism , Protein Domains , Gene Expression , Models, Molecular , Cysteine/chemistry , Cysteine/genetics , Receptors, Scavenger/chemistry , Receptors, Scavenger/genetics , Receptors, Scavenger/metabolism
2.
J Biomol Struct Dyn ; : 1-16, 2024 Jan 17.
Article in English | MEDLINE | ID: mdl-38234051

ABSTRACT

In the recent COVID-19 pandemic, developing effective diagnostic assays is crucial for controlling the spread of the SARS-CoV-2 virus. Multi-domain fusion proteins are a promising approach to detecting SARS-CoV-2 antibodies. In this study, we designed an antigen named CoV2-Pro, containing two RBD domains from SARS-CoV-2 Omicron and Delta variants and one CTD domain of the nucleoprotein in the order of RBD-RBD-N, linked by a super flexible glycine linker. We evaluated the suitability of E. coli Shuffle T7 and BL21 (DE3) strain for expressing CoV2-Pro. Moreover, Bioinformatic studies were conducted first to analyze the tertiary structure of CoV2-Pro. The CoV2-Pro sequences were cloned into a pET-32b (+) vector for expression in E. coli Shuffle T7 and BL21 (DE3). SDS-PAGE and western blot confirmed the protein expression and folding structure. The CoV2-Pro-TRX was purified by Ni-NTA affinity chromatography. Dot blot analysis was performed to evaluate the antigenic characterization of the CoV2-Pro. A molecular docking simulation was conducted to assess the binding affinity of CoV2-Pro with LY-COV555 (Bamlanivimab) monoclonal antibody. A molecular dynamic was performed to analyze the stability of the structure. Bioinformatic and experimental studies revealed a stable conformational 3D structure of the CoV2-Pro. The CoV2-Pro interacted with SARS-CoV-2 antibodies, confirming the correct antigenic structure. We assert with confidence that CoV2-Pro is ideal for developing an ELISA assay for precise diagnosis and rigorous vaccine evaluation during the COVID-19 prevalence.Communicated by Ramaswamy H. Sarma.

3.
Protein J ; 40(6): 907-916, 2021 12.
Article in English | MEDLINE | ID: mdl-34586553

ABSTRACT

Enteropeptidase is a duodenum serine protease that triggers the activation of pancreatic enzymes by remarkably specific cleavages after lysine residues of peptidyl substrate (Asp)4-Lys. This high specific cleavage makes the enzyme a widely used biotechnological tool in laboratory researches and industrial scale. Previous studies both in small and large scales were showed low expression and miss-folding of the expressed protein. In this study, the DNA sequence encoding the light chain (catalytic subunit) of bovine enteropeptidase (EPL) was subcloned into plasmid pET-32b, downstream to the DNA encoding the fusion partner thioredoxin immediately after the EPL cleavage site. SHuffle® T7 Express was selected as an expression host due to the ability to promote proper folding and correction of the mis-oxidized bonds. Expression and purification of protein was performed, and the result of biological activity confirmed that the active EPL was obtained. Optimization of protein expression conditions was accomplished by response surface methodology for significant factors including induction temperature, duration of induction, inducer concentration and OD600 of induction. The best conditions were achieved in 1.05 mM IPTG at OD600 of 0.6 for seven h incubation at 26.5 °C, and a high level of protein expression was obtained in the optimized condition.


Subject(s)
Enteropeptidase , Animals , Catalytic Domain , Cattle , Enteropeptidase/genetics , Enteropeptidase/metabolism , Kinetics , Plasmids
4.
Protein Expr Purif ; 186: 105918, 2021 10.
Article in English | MEDLINE | ID: mdl-34044133

ABSTRACT

Bone morphogenetic protein 2 (BMP21) is a highly interesting therapeutic growth factor due to its strong osteogenic/osteoinductive potential. However, its pronounced aggregation tendency renders recombinant and soluble production troublesome and complex. While prokaryotic expression systems can provide BMP2 in large amounts, the typically insoluble protein requires complex denaturation-renaturation procedures with medically hazardous reagents to obtain natively folded homodimeric BMP2. Based on a detailed aggregation analysis of wildtype BMP2, we designed a hydrophilic variant of BMP2 additionally containing an improved heparin binding site (BMP2-2Hep-7M). Consecutive optimization of BMP2-2Hep-7M expression and purification enabled production of soluble dimeric BMP2-2Hep-7M in high yield in E. coli. This was achieved by a) increasing protein hydrophilicity via introducing seven point mutations within aggregation hot spots of wildtype BMP2 and a longer N-terminus resulting in higher affinity for heparin, b) by employing E. coli strain SHuffle® T7, which enables the structurally essential disulfide-bond formation in BMP2 in the cytoplasm, c) by using BMP2 variant characteristic soluble expression conditions and application of l-arginine as solubility enhancer. The BMP2 variant BMP2-2Hep-7M shows strongly attenuated although not completely eliminated aggregation tendency.


Subject(s)
Bone Morphogenetic Protein 2 , Recombinant Fusion Proteins , Binding Sites/genetics , Bone Morphogenetic Protein 2/chemistry , Bone Morphogenetic Protein 2/genetics , Bone Morphogenetic Protein 2/isolation & purification , Bone Morphogenetic Protein 2/metabolism , Escherichia coli/genetics , Heparin/metabolism , Protein Engineering , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Solubility
5.
Res Pharm Sci ; 15(3): 218-225, 2020 Jun.
Article in English | MEDLINE | ID: mdl-33088322

ABSTRACT

BACKGROUND AND PURPOSE: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with a wide range of therapeutic applications although expression of GM-CSF in Escherichia coli (E. coli) usually leads to formation of insoluble aggregates mostly lack biological activity. The aim of this study was to compare the soluble expression level of GM-CSF in three E. coli strains, BL21 (DE3), SHuffle® T7 and Origami™ 2 (DE3). EXPERIMENTAL APPROACH: The effect of different temperatures and inducer concentrations on soluble expression of GM-CSF was evaluated. The soluble GM-CSF was subjected to endotoxin removal and purification using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, ultrafiltration. The biological activity of produced GM-CSF was evaluated based on its growth promotion effect on TF-1 cell lines by MTT assay method. FINDINGS / RESULTS: A significant improvement of the soluble yield of GM-CSF (about 30% of GM-CSF was expressed as soluble proteins) was observed when protein expression was induced at 30 °C with 0.5 mM isopropyl ß- d-1-thiogalactopyranoside (IPTG) in E. coli Shuffle T7. The soluble GM-CSF with a high purity up to 95 % and specific activity of 1.25 × 104 IU/µg was obtained. CONCLUSION AND IMPLICATIONS: The proposed strategy here can be used to improve the soluble expression of other hard-to-express proteins with similar structural properties (i.e., containing disulfide binds or cysteine).

6.
Iran J Pharm Res ; 17(2): 743-752, 2018.
Article in English | MEDLINE | ID: mdl-29881431

ABSTRACT

Tumor necrosis factor alpha (TNF-α) expression amplifies to excess amounts in several disorders such as rheumatoid arthritis and psoriasis. Although, Anti-TNF biologics have revolutionized the treatment of these autoimmune diseases, formation of anti-drug antibodies (ADA) has dramatically affected their use. The next generation antibodies (e.g. Fab, scFv) have not only reduced resulted immunogenicity, but also proved several benefits including better tumor penetration and more rapid blood clearance. Using affinity selection procedures in this study, a scFv antibody clone was isolated from naïve Tomlinson I phage display library that specifically recognizes and binds to TNF-α. The TNF-α recombinant protein was expressed in genetically engineered Escherichia coli SHuffle® T7 Express, for the first time, which is able to express disulfide-bonded recombinant proteins into their correctly folded states. ELISA-based affinity characterization results indicated that the isolated novel 29.2 kDa scFv binds TNF-α with suitable affinity. In-silico homology modeling study using 'ModWeb' as well as molecular docking study using Hex program confirmed the scFv and TNF-α interactions with a scFv-TNF- α binding energy of around -593 kj/mol which is well in agreement with our ELSIA results. The cloned scFv antibody may be potentially useful for research and therapeutic applications in the future.

7.
Int J Biol Macromol ; 99: 173-178, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28215564

ABSTRACT

TNF-α, a prototype member of the TNF family of ligands, has both pro-inflammatory and immune-regulatory functions, which make it as an appropriate therapeutic target for selective blockade in antibody therapy of many diseases like in rheumatoid arthritis. Using two models of SHuffle® T7 Express and BL21 (DE3) cells, we have expressed this protein and recognized it by SDS-PAGE analysis. FTIR biospectroscopy of the resulted purified proteins has been performed and mathematical calculations has been done for further identification of the structural and conformational differences between the two products. Our results showed some differences in disulfide bond formation and ß-sheet turns between these two recombinant proteins. To the best of our knowledge, this is the first study that compare secondary structure of recombinant proteins in both conventional and next generation Escherichia coli based expression systems using reliable, simple, rapid and economic ATR-FTIR analysis. Whether these differences might have significant effects on TNF-α inflammatory and immune-regulatory function in biological systems might be very much important and need further investigations.


Subject(s)
Escherichia coli/cytology , Escherichia coli/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spectroscopy, Fourier Transform Infrared , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/genetics , Cloning, Molecular , Gene Expression , Humans , Protein Structure, Secondary , Recombinant Proteins/isolation & purification , Tumor Necrosis Factor-alpha/isolation & purification
8.
Bioengineering (Basel) ; 3(4)2016 Nov 21.
Article in English | MEDLINE | ID: mdl-28952595

ABSTRACT

The glycosyltransferase HisDapGalNAcT2 is the key protein of the Escherichia coli (E. coli) SHuffle® T7 cell factory which was genetically engineered to allow glycosylation of a protein substrate in vivo. The specific activity of the glycosyltransferase requires time-intensive analytics, but is a critical process parameter. Therefore, it has to be monitored closely. This study evaluates fluorometric in situ monitoring as option to access this critical process parameter during complex E. coli fermentations. Partial least square regression (PLS) models were built based on the fluorometric data recorded during the EnPresso® B fermentations. Capable models for the prediction of glucose and acetate concentrations were built for these fermentations with rout mean squared errors for prediction (RMSEP) of 0.19 g·L-1 and 0.08 g·L-1, as well as for the prediction of the optical density (RMSEP 0.24). In situ monitoring of soluble enzyme to cell dry weight ratios (RMSEP 5.5 × 10-4 µg w/w) and specific activity of the glycosyltransferase (RMSEP 33.5 pmol·min-1·µg-1) proved to be challenging, since HisDapGalNAcT2 had to be extracted from the cells and purified. However, fluorescence spectroscopy, in combination with PLS modeling, proved to be feasible for in situ monitoring of complex expression systems.

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