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1.
Int J Mol Sci ; 22(14)2021 Jul 08.
Article in English | MEDLINE | ID: mdl-34298982

ABSTRACT

Engineering of biomimetic motives have emerged as promising approaches to improving cells' binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface's receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5ß1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.


Subject(s)
Affinity Labels , Biomimetic Materials , Fibronectins , Oligopeptides , Peptide Fragments , Recombinant Fusion Proteins/chemistry , Adsorption , Cell Adhesion , Coated Materials, Biocompatible , Escherichia coli , Extracellular Matrix/chemistry , Genetic Vectors , Integrin alpha5beta1/metabolism , Ligands , Mass Spectrometry , Polystyrenes , Protein Binding , Protein Domains , Protein Engineering , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism
2.
Materials (Basel) ; 14(11)2021 Jun 03.
Article in English | MEDLINE | ID: mdl-34205116

ABSTRACT

In this study, ultraporous aluminas (UPA) were synthesized as new effective adsorbents for Remazol Brilliant Blue R (RBBR) removal from aqueous solutions. The UPA monoliths were grown via facile oxidation process, followed by isochronous annealing treatment in air at different temperatures, through which γ, θ, and α phase polycrystalline fibrous grains of UPA can be accordingly obtained. The experimental factors that affect the material adsorption performances including initial pH, contact time, and temperature were comprehensively studied by batch experiments. The RBBR adsorption isotherms of UPA(γ) and UPA(θ) powders were found almost identical, while UPA(α) powders showed low effectiveness. To obtain the desirable mechanical stability of the UPA monolith with considerable RBBR adsorption capacity, UPA(θ) powders were further studied. The UPA(θ) powders exhibited maximum RBBR adsorption at pH 2 due to the positively charged surface under acidic conditions. Compared with the Lagergren pseudo-first-order model, the pseudo-second-order model was found to explain the adsorption kinetics better. Despite the film diffusion dominating the adsorption process, the contributions of the intraparticle diffusion and chemical reactions were also found significant. The adsorption equilibrium data at different temperatures were fitted by the Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich (D-R) isotherm models. The Langmuir model was found the most effective in the description of equilibrium data, and the maximum RBBR adsorption capacity retained by UPA(θ) powders was 122.55 mg·g-1 at 295 K. Thermodynamic parameters (ΔG0, ΔH0, and ΔS0) indicated the adsorption process was spontaneous and exothermic in nature.

3.
Mol Biotechnol ; 55(3): 227-35, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23780701

ABSTRACT

In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin-agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.


Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Escherichia coli/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Animals , Bacterial Proteins , Carrier Proteins/chemistry , Carrier Proteins/genetics , DNA-Directed DNA Polymerase/biosynthesis , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/isolation & purification , Escherichia coli/metabolism , Genetic Vectors , Heme-Binding Proteins , Hemeproteins/chemistry , Histidine/chemistry , Histidine/genetics , Rats , Recombinant Fusion Proteins/chemistry , Sepharose/analogs & derivatives , Vesicular Transport Proteins/biosynthesis , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/isolation & purification
4.
J Biotechnol ; 158(3): 97-103, 2012 Apr 15.
Article in English | MEDLINE | ID: mdl-22300512

ABSTRACT

The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins. The difficulty in obtaining this enzyme led us to look for an optimal method for its use. In this work, we produced both the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence (Streptag II-TEV(WT), and Streptag II-TEV(S219V), respectively). The two enzymes were affinity immobilized on a streptavidin-agarose matrix and compared to their respective free forms. Both immobilized Streptag II-TEV(WT) and Streptag II-TEV(S219V) were active on the 74-kDa Streptag II substrate with a retained activity of 83.5% and 81%, respectively compared to their free corresponding forms. The slight enzyme activity decrease caused by the immobilization was balanced by the enhanced stability and the successful repetitive use of the proteolytic columns. Thus, the wild-type and the mutant immobilized proteases were used, during a period of 18 months, for nine batch reactions with retention of 38% and 51% of their initial activities, respectively. The present results demonstrate that immobilized TEV protease on streptavidin-agarose is an attractive and efficient tool for fusion protein cleavage, especially when the target protein is fused to a streptagged fusion partner. Using this strategy, the total process can be shortened by performing the cleavage and the recovery of the purified target protein in one step.


Subject(s)
Endopeptidases/chemistry , Enzymes, Immobilized/chemistry , Plant Viruses/enzymology , Proteolysis , Recombinant Fusion Proteins/chemistry , Viral Proteins/chemistry , Endopeptidases/biosynthesis , Endopeptidases/genetics , Enzymes, Immobilized/genetics , Enzymes, Immobilized/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Plant Viruses/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Streptavidin/chemistry , Streptavidin/genetics , Streptavidin/metabolism , Viral Proteins/biosynthesis , Viral Proteins/genetics
5.
Protein Expr Purif ; 2011 Sep 02.
Article in English | MEDLINE | ID: mdl-21889987

ABSTRACT

The Tobacco Etch Virus (TEV) protease is frequently used in the cleavage of recombinant fusion proteins because of its efficiency and high specificity. In this work, we present a new recombinant form of TEV termed Streptag II-TEV for high-level production and purification of TEV protease from Escherichia coli and compare it to the hexahistidine (6xHis) tagged version of TEV. The effects of varying the host strain, the bacterial induction temperature (25, 30 and 37°C) and the IPTG inducer concentration on production and solubility of the two recombinant TEV proteases have been examined. Optimal Streptag II-TEV protein expression were obtained in the E. coli KRX strain under an induction temperature of 25°C in the presence of IPTG at 0.5mM. In these conditions, soluble Streptag II-TEV and 6xHis-TEV proteases accounted for about 25% and 18% of total soluble proteins, respectively. About 70% of Streptag II-TEV and 60% of 6xHis-TEV were detected in the supernatant. Streptag II-TEV protease purifies to near homogeneity (approximately 99%) via a simple, single step Strep-Tactin chromatography purification protocol based on the presence of Streptag II. The higher production of Streptag II-TEV coupled to its purification and cleavage efficiencies make it an attractive alternate to 6xHis-TEV.

6.
Protein Expr Purif ; 75(1): 75-82, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20817099

ABSTRACT

The Tobacco Etch Virus (TEV) protease is frequently used in the cleavage of recombinant fusion proteins because of its efficiency and high specificity. In this work, we present a new recombinant form of TEV termed Streptag II-TEV for high-level production and purification of TEV protease from Escherichia coli and compare it to the hexahistidine (6xHis) tagged version of TEV. The effects of varying the host strain, the bacterial induction temperature (25, 30 and 37°C) and the IPTG inducer concentration on production and solubility of the two recombinant TEV proteases have been examined. Optimal Streptag II-TEV protein expression were obtained in the E. coli KRX strain under an induction temperature of 25°C in the presence of IPTG at 0.5 mM. In these conditions, soluble Streptag II-TEV and 6xHis-TEV proteases accounted for about 25% and 18% of total soluble proteins, respectively. About 70% of Streptag II-TEV and 60% of 6xHis-TEV were detected in the supernatant. Streptag II-TEV protease purifies to near homogeneity (approximately 99%) via a simple, single step Strep-Tactin chromatography purification protocol based on the presence of Streptag II. The higher production of Streptag II-TEV coupled to its purification and cleavage efficiencies make it an attractive alternate to 6xHis-TEV.


Subject(s)
Cloning, Molecular , Endopeptidases/genetics , Escherichia coli/genetics , Histidine/genetics , Oligopeptides/genetics , Plant Viruses/enzymology , Viral Proteins/genetics , Base Sequence , Endopeptidases/isolation & purification , Endopeptidases/metabolism , Gene Expression , Genetic Vectors/genetics , Molecular Sequence Data , Plant Viruses/genetics , Plasmids/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Nicotiana/virology , Viral Proteins/isolation & purification , Viral Proteins/metabolism
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