Recent advances in covalent, site-specific protein immobilization

Abstract

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. Covalent, site-specific immobilization strategies are robust and can provide the level of control that is desired in this kind of application. Recent advances include the use of enzymes, such as sortase A, to couple proteins in a site-specific manner to materials such as microbeads, glass, and hydrogels. Also, self-labeling tags such as the SNAP-tag can be employed. Last but not least, chemical approaches based on bioorthogonal reactions, like the azide–alkyne cycloaddition, have proven to be powerful tools. The lack of comparative studies and quantitative analysis of these immobilization methods hampers the selection process of the optimal strategy for a given application. However, besides immobilization efficiency, the freedom in selecting the site of conjugation and the size of the conjugation tag and the researcher’s expertise regarding molecular biology and/or chemical techniques will be determining factors in this regard.

Generation of Multifunctional Magnetic Nanoparticles with Amplified Catalytic Activities by Genetic Expression of Enzyme Arrays on Bacterial Magnetosomes

“…Magnetic imaging techniques for instance would benefit from multimodal particles that in addition to their magnetic and fluorescent properties display enzyme moieties, receptors, or antibodies on the surface. In addition, the properties of magnetic biosensors and protein arrays for drug screening are based on the efficient immobilization of (enzyme) proteins, and a high degree of control over the immobilization process is required, [51,52] which makes genetically engineered magnetosomes to an attractive platform for the high-level display of foreign proteins.…”

Purification‐Free, Target‐Selective Immobilization of a Protein from Cell Lysates

“…As a first step for direct immobilization of sfGFP on solid supports, the AzF containing an azide group was introduced into a specific site of sfGFP. As an AzF incorporation site, a position between 214 and 215 was chosen, because it is distant from the sfGFP chromophore and has a relatively high solvent accessibility facilitating efficient conjugation . Therefore, an amber codon was introduced into position 215 of sfGFP.…”

“…Conventional covalent attachment techniques using primary amines or sulfhydryl groups in a target protein usually resulted in poor selectivity toward a target protein, poor orientation control, and significant activity loss due to chemical modification at critical sites . Emerging site‐specific immobilization techniques addressed some of these issues . Certain enzymes, such as farnesyltransferase and N‐myristoyltransferase, were used to incorporate a bioorthogonal chemical handle into a specific site of a target protein, followed by immobilization of a target protein onto supports .…”

3D Laser Micro‐ and Nanoprinting: Challenges for Chemistry

“…However, to extend it to create more complex environments and direct cell differentiation, proteins with the orthogonal linkers (e.g. His, SNAP, Halo, or CLIP tags) are required . These tagged proteins are usually not commercially available and, therefore, need to be specifically expressed and purified.…”