Abstract
Protein modifications through genetic code engineering have a remarkable impact on macromolecule engineering, protein translocation, protein–protein interaction, and cell biology. We used the newly developed molecular biology approach, genetic code engineering, for fine-tuning of proteins for biological availability. Here, we have introduced 3, 4-dihydroxy-l-phenylalanine in recombinant proteins by selective pressure incorporation method for protein-based cell labeling applications. The congener proteins treated with tyrosinase convert 3, 4-dihydroxy-l-phenylalanine to dopaquinone for strain-promoted click chemistry. Initially, the single-step Strain-Promoted Oxidation-Controlled Cyclooctyne-1,2-quinone Cycloaddition was studied using tyrosinase catalyzed congener protein and optimized the temporally controlled conjugation with (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol. Then, the feasibility of tyrosinase-treated congener annexin A5 with easily reactive quinone functional moiety was conjugated with fluorescent tag dibenzocyclooctyne-PEG4-TAMRA for labeling of apoptotic cells. Thus, the congener proteins-based products demonstrate selective cell labeling and apoptosis detection in EA.hy926 cells even after the protein modifications. Hence, genetic code engineering can be coupled with click chemistry to develop various protein-based fluorescent labels.
Highlights
Protein modifications through chemical and genetic methods have a remarkable impact on cell labeling, bio-conjugation studies, protein translocation, protein-protein interaction [1,2]
DOPA was treated with 60 U of tyrosinase to produce dopaquinone intermediate in tris buffer pH 6.8, which is further converted into dopachrome within a second (t1/2>1s) by an intramolecular cyclization (Michael- 1, 4 additions) and forms stable melanin pigment [30, 31]
A sharp absorbance reduction represents a straight line in the UV/Vis spectral region (200-600 nm) which was obtained after the tyrosinase-treated DOPA conjugated with BCN (Fig. 1b), and this indicates that the reaction is stabilized after the addition of BCN
Summary
Protein modifications through chemical and genetic methods have a remarkable impact on cell labeling, bio-conjugation studies, protein translocation, protein-protein interaction [1,2]. The chemo-selective modification of canonical amino acids on proteins had initially been executed via amide coupling of lysine [3], sulfhydryl reaction of cysteine [4], N-terminal transamination [5] and, oxidative coupling of proline without alteration in the protein structure and function [6]. Most of the chemical modifications have limitations in biocompatibility, regioselectivity, and homogeneity [7]. Despite these challenges, in recent decades’ genetic incorporation of non-canonical amino acids [8] for protein modification has extended the possibilities of proteins for novel applications. The unprecedented specificity of these enzymes has enabled modification of the full-length proteins with fluorophores through expanding the genetic code
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