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

Abstract

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization).

Fusion of Chitin-Binding Domain From Chitinolyticbacter meiyuanensis SYBC-H1 to the Leaf-Branch Compost Cutinase for Enhanced PET Hydrolysis

“…Protein concentrations were determined by the Bradford method ( Campion et al, 2017 ). The amount of adsorbed ChBD-GFP was determined using the fluorescence intensity detected by Hitachi F7000 fluorescence spectrophotometer similarly as describe before ( Cha and Kwon, 2018 ). The adsorption rate was calculated with the formula below: …”

Immobilization of Proteins with Controlled Load and Orientation

“…Unfortunately, the majority of genetically encodable bioorthogonal reactions available for protein immobilization proceed at sluggish reaction rates or require conditions that compromise protein functionality. For example, the commonly used strain-promoted azide–alkyne click reaction proceeds at a rate of ∼0.1–10 M –1 s –1 which necessitates high protein concentrations and overnight incubations that tend to promote nonspecific protein adsorption, protein aggregation, and loss in enzyme activity, despite its widespread use. , The use of a copper catalyst can increase the azide–alkyne click reaction ,,, rate by 10–100 fold, but this still results in a reaction half-life of ∼3 h at 1 μM concentration of both reagents . Worse yet, the reactive oxygen species generated by copper-catalyst azide–alkyne click reactions have been well documented to affect the structure and functional integrity of proteins, making it impossible to predict the quantity of functional protein immobilized with this approach. , …”

Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1

“…With regards to ChBD-GFP and ChBD-LGOX, lysates were used directly for adsorption under the optimal condition and the absorption effects were detected by SDS-PAGE. The absorption rate of ChBD-GFP was characterized by measuring the quality of GFP which was converted from the fluorescence intensity (Cha and Kwon, 2018) and absorption rate here was calculated as the above formula. The fluorescence intensity was detected by LGOX activity in absorbent Initial LGOX activity × 100%.…”