Synthesis of Na2S2O4 mediated cleavable affinity tag for labeling of O-GlcNAc modified proteins via azide-alkyne cycloaddition

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.

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Conformational Studies on Modified Proteins and Peptides

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)62826-1 ◽

1970 ◽

Vol 245 (19) ◽

pp. 5122-5128

Author(s):

M.Z. Atassi ◽

R.P. Singhal

Keyword(s):

Conformational Studies ◽

Modified Proteins ◽

Proteins And Peptides

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The Alterations in Methylene Blue/Light-Treated Frozen Plasma Proteins Revealed by Proteomics

Transfusion Medicine and Hemotherapy ◽

10.1159/000515119 ◽

2021 ◽

pp. 1-8

Author(s):

Tiange Wu ◽

Xiaoning Wang ◽

Kai Ren ◽

Xiaochen Huang ◽

Jiankai Liu

Keyword(s):

Mass Spectrometry ◽

Methylene Blue ◽

Western Blot ◽

Blue Light ◽

Differentially Expressed ◽

Enzyme Linked Immunosorbent Assay ◽

Differentially Expressed Protein ◽

Significant Difference ◽

Modified Proteins ◽

Frozen Plasma

Introduction: The aim of this study was to investigate the modified proteins in methylene blue/light-treated frozen plasma (MB-FP) compared with fresh frozen plasma (FFP) in order to gain a better application of MB/light-treated plasma in clinic transfusion. Methods: MB-FP and FFP were collected from Changchun central blood station, and a trichloroacetic acid/acetone precipitation method was used to remove albumin for the enrichment of lower abundance proteins. The plasma protein in MB-FP and FFP were separated using two-dimensional gel electrophoresis (2-DE) and the differentially expressed protein spots were analyzed using mass spectrometry. Finally, the differentially expressed proteins were tested using Western blot and enzyme-linked immunosorbent assay (ELISA). Results: Approximately 14 differentially expressed protein spots were detected in the MB-FP, and FFP was chosen as the control. After 2-DE comparison analysis and mass spectrometry, 8 significantly differentially expressed protein spots were identified, corresponding to 6 different proteins, including complement C1r subcomponent (C1R), inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4), keratin, type II cytoskeletal 1 (KRT1), hemopexin (HPX), fibrinogen gamma chain (FGG), and transthyretin (TTR). Western blot showed no significant difference in the expression level of KRT1 between MB-FP and FFP (p > 0.05). Both Western blot and ELISA indicated that the level of HPX was significantly higher in FFP than in MB-FP (p < 0.05). Conclusion: This comparative proteomics study revealed that some significantly modified proteins occur in MB-FP, such as C1R, ITI-H4, KRT1, HPX, FGG, and TTR. Our findings provide more theoretical data for using MB-FP in transfusion medicine. However, the relevance of the data for the transfusion of methylene blue/light-treated plasma remains unclear. The exact modification of these proteins and the effects of these modified proteins on their functions and their effects in clinical plasma infusion need to be further studied.

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Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

International Journal of Molecular Sciences ◽

10.3390/ijms22147362 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7362

Author(s):

Amina Ben Abla ◽

Guilhem Boeuf ◽

Ahmed Elmarjou ◽

Cyrine Dridi ◽

Florence Poirier ◽

...

Keyword(s):

Specific Binding ◽

Cellular Adhesion ◽

High Yield ◽

Adhesion Assay ◽

Cell Binding ◽

Affinity Tag ◽

Binding Domains ◽

Rational Development ◽

Fibronectin Type Iii ◽

Arginine Glycine Aspartic Acid

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 α5β1 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.

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