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Total Chemical Synthesis of a Functionalized GFP Nanobody.

ABSTRACT: Chemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs with a range of chemical modifications such as tags, linkers, reporter groups, and subsequently, Nb-drug conjugates. Herein, we describe the total chemical synthesis of a 123 amino-acid Nb against GFP. A native chemical ligation- desulfurization strategy was successfully applied for the synthesis of this GFP Nb, modified with a propargyl (PA) moiety for on-demand functionalization. Biophysical characterization indicated that the synthetic GFP Nb-PA was correctly folded after internal disulfide bond formation. The synthetic Nb-PA was functionalized with a biotin or a sulfo-cyanine5 dye by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), resulting in two distinct probes used for functional in vitro validation in pull-down and confocal microscopy settings.

SUBMITTER: Huppelschoten Y

PROVIDER: S-EPMC9804225 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Total Chemical Synthesis of a Functionalized GFP Nanobody.

Huppelschoten Yara Y Elhebieshy Angela F AF Hameed Dharjath S DS Sapmaz Aysegul A Buchardt Jens J Nielsen Thomas E TE Ovaa Huib H van der Heden van Noort Gerbrand J GJ

Chembiochem : a European journal of chemical biology 20220823 19

Chemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs with a range of chemical modifications such as tags, linkers, reporter groups, and subsequently, Nb-drug conjugates. Herein, we describe the total chemical synthesis of a 123 amino-acid Nb against GFP. A native chemical ligation- desulfurization strategy was successfully applied for the synthesis of this ...[more]

PMID: 35920208

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Project description:The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2-6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8-33% overall yield with 90-98% purity despite the omission of HPLC purification.

Dataset Information

Total Chemical Synthesis of a Functionalized GFP Nanobody.

Publications

Total Chemical Synthesis of a Functionalized GFP Nanobody.

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