Immobilization of Proteins with Controlled Load and Orientation

Abstract

Biomaterials based on immobilized proteins are key elements of many biomedical and industrial technologies. However, applications are limited by an inability to precisely construct materials of high homogeneity and defined content. We present here a general "protein-limited immobilization" strategy by combining the rapid, bioorthogonal, and biocompatible properties of a tetrazine-strained trans-cyclooctene reaction with genetic code expansion to sitespecifically place the tetrazine into a protein. For the first time, we use this strategy to immobilize defined amounts of oriented proteins onto beads and flat surfaces in under 5 min at submicromolar concentrations without compromising activity. This approach opens the door to generating and studying diverse protein-based biomaterials that are much more precisely defined and characterized, providing a greater ability to engineer properties across a wide range of applications.

Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

“…The IEDDA reaction is a [4+2] cycloaddition that occurs between an electron-deficient diene such as a 1,2,4,5-tetrazine and an electron-rich dienophile such as a strained alkene 1 . Bioorthogonal IEDDA reactions, such as those first described by Fox and colleagues, can reach rates upwards of 10 6 M -1 s -1 , allowing complete reaction within minutes at sub-micromolar concentrations 1,9,[11][12][13][14] . Amino acid derivatives containing azide, cyclopropene, alkyne, trans-cyclooctene (TCO), and tetrazine functionalities have all been genetically encoded into proteins 15 , albeit not yet in a manner conducive to dual encoding and subsequent intracellular labeling in vivo.…”

Faster Surface Ligation Reactions Improve Immobilized Enzyme Structure and Activity

“…As shown in Figure 1A, these constructs, referred to hereafter as CA AzF , CA Cys , and CA Tet , were designed to permit site-specific ligation to either DBCO, maleimide, or sTCO-functionalized silica microparticles (1 μm mean diameter), via slow, intermediate or fast ligation reaction rate constant, respectively. Residue position 126 was chosen because it is remote from the active site, which would reduce the probability that immobilization would result in obstruction of the active site 12 . Importantly, to generate CA AzF , CA Cys , and CA Tet , we used the thermally stable variant C205S in which the only native cysteine in CA was replaced with a serine.…”

“…Cloning, Expression, and Purification of CA constructs CA constructs (Table S1) were cloned from a C205S thermal stable variant using methods that were previously described elsewhere 12 . Following cloning, CA constructs were produced using the DEAL approach as we've previously described 35 .…”

“…Additionally, the promise of such biohybrid materials is frequently limited by the fact that enzymes generally function in only a narrow range of conditions. As such, the development of strategies to improve the functional utility of enzymes upon immobilization has been the focus of considerable effort [9][10][11][12] .…”

Predicting Protein Function and Orientation on a Gold Nanoparticle Surface Using a Residue-Based Affinity Scale

“…Understanding the behavior of surface-adsorbed proteins on nanoparticles (NP) is crucial when NPs are used in vivo as a therapeutic drug carrier, phototherapeutic agent, or imaging tool [1][2][3][4] . There is increasing evidence that the composition of the adsorbed proteins is not as important as their structure and orientation [5][6][7] , as the latter governs the three-dimensional presentation of proteins for molecular recognition, which will define the biological performance and fate of the NP [8][9][10] . For example, the arrangement and orientation of the adsorbed proteins on a NP define the distribution of epitopes presented for immune response 5 and determine the success of cell targeting 6,9 .…”