Simultaneous “One Pot” Expressed Protein Ligation and CuI‐Catalyzed Azide/Alkyne Cycloaddition for Protein Immobilization

  title={Simultaneous “One Pot” Expressed Protein Ligation and CuI‐Catalyzed Azide/Alkyne Cycloaddition for Protein Immobilization},
  author={Max Steinhagen and Kai Holland‐Nell and Morten Meldal and Annette G. Beck‐Sickinger},
Proteins, enzymes in particular, are becoming more and more important in synthesis and the creation of stereoand regioselectively novel products with high specificity. They also play an important role in the development of diagnostic systems, microarrays and biosensors. The anchoring of proteins to solid supports can facilitate these systems, but to maintain the native properties and homogeneity, site specific immobilization is preferred, which in turn calls for site specific introduction of… 

Recent advances in covalent, site-specific protein immobilization

The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support.

Recent advances in covalent, site-specific protein

  • Biology, Chemistry
  • 2019
The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support.

Advances in Merging Triazoles with Peptides and Proteins

Five-membered heterocycles have found extensive use as peptide- and disulfide-bond mimics in peptidomimetics and recent developments of new ligands and catalysts for the CuAAC reaction have contributed to the promising possibilities that triazoles provide for future applications in the peptide and protein field.

Facile chemical functionalization of proteins through intein-linked yeast display.

This work sought to simultaneously release yeast surface-displayed proteins and selectively conjugate with chemical functionalities compatible with EPL and click chemistry to eliminate the need for soluble protein expression and purification.

Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates

The sequence of immobilization via hydrazone ligation, on‐surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield, and the synthetic proteins were functional as demonstrated by an on‐ surface fluorescence‐based saturation binding analysis.

An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation.

The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies and paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling.

Preparation of C-terminally modified chemokines by expressed protein ligation.

A general protocol for the preparation of C-terminally modified SDF-1α including tips and tricks for practical work is provided and it is believed that this protocol can be easily adapted to other chemokines and many proteins in general.

Semi-synthesis of chemokines.



Diels-Alder ligation of peptides and proteins.

The results demonstrate that the Diels-Alder ligation offers an advantageous and technically straightforward new opportunity for the site-specific equipment of peptides and proteins with further functional groups and labels.

Self-assembled monolayers with latent aldehydes for protein immobilization.

A general modular strategy was introduced by which SAM components with vicinal diol functions were rapidly synthesized with high yield, thereby producing latent aldehyde-containing SAM components for metal oxides and hydrogen-terminated silicon, respectively, to show the general usefulness of the new synthetic design.

Protein and electrode engineering for the covalent immobilization of P450 BMP on gold.

Results from tapping mode atomic force microscopy show that the double mutant and the C62S did not lead to stably immobilized P450 protein, confirming the necessity of the solvent exposed C62.

Semisynthesis of proteins by expressed protein ligation.

  • T. Muir
  • Biology, Chemistry
    Annual review of biochemistry
  • 2003
In this review, the development of this technology is discussed, its broad application to biological systems, and its possible role in the area of proteomics are discussed.

Synthesis of N-terminally linked protein dimers and trimers by a combined native chemical ligation-CuAAC click chemistry strategy.

A novel method for the synthesis of N-terminally linked protein multimers is reported and the orthogonal nature of this reaction allows the production of protein heteromultimers, and this is demonstrated by synthesis of a protein heterodimer.

An intein-mediated site-specific click conjugation strategy for improved tumor targeting of nanoparticle systems.

This new EPL-click conjugation strategy is applied to create superparamagnetic iron oxide nanoparticles (SPIO) labeled with HER2/neu affibodies, which prove to be highly potent and receptor-specific in both in vitro cell studies and murine tumor models.

Oriented immobilization of farnesylated proteins by the thiol-ene reaction.

This method enables the oriented covalent immobilization of proteins directly from expression lysates without additional purification or derivatization steps.

Click Chemistry: Diverse Chemical Function from a Few Good Reactions.

The stringent criteria for a process to earn click chemistry status are described along with examples of the molecular frameworks that are easily made using this spartan, but powerful, synthetic strategy.

Specifically Immobilised Aldo/Keto Reductase AKR1A1 Shows a Dramatic Increase in Activity Relative to the Randomly Immobilised Enzyme

Investigation of the kinetic parameters revealed remarkably improved activity for the site‐specifically immobilised enzyme, which was comparable to that of the wild‐type enzyme in solution and 60–300‐fold greater than that ofThe randomly immobilized enzymes.

A general method for site-specific incorporation of unnatural amino acids into proteins.

The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.