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Total chemical synthesis of photoactivatable proteins for light-controlled manipulation of antigen-antibody interactions.

ABSTRACT: We report the chemical synthesis of the first photo-activatable protein antigen that can be used to study antigen-antibody interaction mediated responses in B cells. This strategy facilitated fine tuning of the caged protein antigen to optimize its bioactivity and photochemical properties. One optimal molecule, HEL-K96NPE, was totally inert to hen egg lysozyme (HEL)-specific B cells and could only restore its antigenicity upon photoactivation. Combined with real time live cell imaging, the utility of HEL-K96NPE was demonstrated as a proof of concept to quantify B cell synapse formation and calcium influx responses at the single cell level.

SUBMITTER: Tang S

PROVIDER: S-EPMC5965250 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Total chemical synthesis of photoactivatable proteins for light-controlled manipulation of antigen-antibody interactions.

Tang Shan S Wan Zhengpeng Z Gao Yiren Y Zheng Ji-Shen JS Wang Jing J Si Yan-Yan YY Chen Xin X Qi Hai H Liu Lei L Liu Wanli W

Chemical science 20151211 3

We report the chemical synthesis of the first photo-activatable protein antigen that can be used to study antigen-antibody interaction mediated responses in B cells. This strategy facilitated fine tuning of the caged protein antigen to optimize its bioactivity and photochemical properties. One optimal molecule, HEL-K<sub>96</sub>NPE, was totally inert to hen egg lysozyme (HEL)-specific B cells and could only restore its antigenicity upon photoactivation. Combined with real time live cell imaging ...[more]

PMID: 29899912

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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.

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Total chemical synthesis of photoactivatable proteins for light-controlled manipulation of antigen-antibody interactions.

Publications

Total chemical synthesis of photoactivatable proteins for light-controlled manipulation of antigen-antibody interactions.

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