Site-Specific Incorporation of p-Propargyloxyphenylalanine in a Cell-Free Environment for Direct Protein−Protein Click Conjugation

Abstract

The tyrosine analog p-propargyloxyphenylalanine (pPa), like tyrosine, has limited water solubility. It has been postulated that this limited solubility has contributed to reduced cellular uptake of pPa and thus reduced in vivo incorporation of pPa into proteins. Using a cell-free protein synthesis system (CFPS) to circumvent cellular uptake, pPa has been incorporated site-specifically into proteins with high specificity at yields up to 27 times greater than the highest previously reported yield. The alkyne group present on proteins incorporated with pPa provides a reactive residue for site-specific bioconjugation with the copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition (CuAAC). Previously, incorporation of another CuAAC-compatible unnatural amino acid p-azido-l-phenylalanine (pAz) was demonstrated with CFPS. However, incorporation of pPa may be preferred over pAz due to the instability of the pAz's aryl-azido moiety upon UV or near-UV light exposure. Also, the ability to incorporate site-specifically both reactants of the CuAAC (the alkyne group of pPa and the azido group of pAz) into proteins enables direct site-specific conjugation of heterologous proteins. We have demonstrated (for the first time to our knowledge) a one-step, linker-less, site-specific, direct protein-to-protein conjugation using CuAAC and unnatural amino acids.

Efficient disulfide bond formation in virus-like particles

“…The Q␤ coat protein gene was expression optimized and synthesized from custom oligonucleotides. The gene was inserted into the pY71 plasmid (Bundy and Swartz, 2010) and expressed with the SS-CFPS protocol resulting in yields of 498 ± 42 g/mL total protein and 442 ± 48 g/mL of soluble protein (n = 3; ± SD). After velocity sedimentation, the assembly efficiency was determined to be only 5% or 26 g/mL based on the expected location of assembled Q␤ VLP (supplementary data).…”

“…To address this concern, we have reported a high yielding Esherichia coli-based cell-free protein synthesis (CFPS) system (Bundy et al, 2008) (Scheme 1). CFPS is a highly adaptable system due to its open transcription/translation environment where the pH, ionic strength, chemical composition, redox potential, chaperone concentration, and production rate can be directly controlled enabling the production of a wide range of proteins from eukaryotic and prokayotic organisms (Bundy and Swartz, 2010;Wuu and Swartz, 2008). In addition, the resources Scheme 1.…”

Synthesis of Heterobifunctional Protein Fusions Using Copper‐Free Click Chemistry and the Aldehyde Tag

“…In this direction, Bundy and Swartz have implemented cell-free protein synthesis to install azide and alkyne amino acids into green fluorescent protein for Cu-catalyzed dimerization. [22] This approach, however, suffered from low protein expression as well as Cu-induced protein damage.…”

Preparation of unnatural N-to-N and C-to-C protein fusions

“…Structural analogs of ubiquitin dimers were prepared by a combination of intein-based ligation, site-specific mutation, and copper-catalyzed click chemistry (22,23). Site-specific incorporation of propargyloxyphenylalanine facilitated the synthesis of green fluorescent protein (GFP) dimers (19,24).Nonetheless, the synthesis of bispecifics would benefit from a method that is orthogonal to the published methods and that allows easy access to modified native protein, as well as enables efficient nonnatural conjugation of protein termini. Moreover, the availability of orthogonal methods will make possible the synthesis of protein structures of even greater complexity (heterotrimers and higher-order complexes).…”

“…Structural analogs of ubiquitin dimers were prepared by a combination of intein-based ligation, site-specific mutation, and copper-catalyzed click chemistry (22,23). Site-specific incorporation of propargyloxyphenylalanine facilitated the synthesis of green fluorescent protein (GFP) dimers (19,24).…”