Dataset Information

Site-Selective Cysteine-Cyclooctyne Conjugation.

ABSTRACT: We report a site-selective cysteine-cyclooctyne conjugation reaction between a seven-residue peptide tag (DBCO-tag, Leu-Cys-Tyr-Pro-Trp-Val-Tyr) at the N or C?terminus of a peptide or protein and various aza-dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO-tag increases the rate of the thiol-yne reaction 220-fold, thereby enabling selective conjugation of DBCO-tag to DBCO-linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO-tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. This study demonstrates that short peptide tags can aid in accelerating bond-forming reactions that are often slow to non-existent in water.

SUBMITTER: Zhang C

PROVIDER: S-EPMC6150453 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Site-Selective Cysteine-Cyclooctyne Conjugation.

Zhang Chi C Dai Peng P Vinogradov Alexander A AA Gates Zachary P ZP Pentelute Bradley L BL

Angewandte Chemie (International ed. in English) 20180426 22

We report a site-selective cysteine-cyclooctyne conjugation reaction between a seven-residue peptide tag (DBCO-tag, Leu-Cys-Tyr-Pro-Trp-Val-Tyr) at the N or C terminus of a peptide or protein and various aza-dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO-tag increases the rate of the thiol-yne reaction 220-fold, thereby enabling selective conjugation of DBCO-tag to DBCO-linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO-ta ...[more]

PMID: 29575377

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Project description:Site-selective conjugation generally requires both (i) molecular engineering of the protein of interest to introduce a conjugation site at a defined location and (ii) a site-specific conjugation technology. Three N-terminal interferon α2-a (IFN) variants with truncated histidine tags were prepared and conjugation was examined using a bis-alkylation reagent, PEG(10kDa)-mono-sulfone 3. A histidine tag comprised of two histidines separated by a glycine (His2-tag) underwent PEGylation. Two more IFN variants were then prepared with the His2-tag engineered at different locations in IFN. Another IFN variant was prepared with the His-tag introduced in an α-helix, and required three contiguous histidines to ensure that two histidine residues in the correct conformation would be available for conjugation. Since histidine is a natural amino acid, routine methods of site-directed mutagenesis were used to generate the IFN variants from E. coli in soluble form at titres comparable to native IFN. PEGylation conversions ranged from 28-39%. A single step purification process gave essentially the pure PEG-IFN variant (>97% by RP-HPLC) in high recovery with isolated yields ranging from 21-33%. The level of retained bioactivity was strongly dependent on the site of PEG conjugation. The highest biological activity of 74% was retained for the PEG10-106(HGHG)-IFN variant which is unprecedented for a PEGylated IFN. The His2-tag at 106(HGHG)-IFN is engineered at the flexible loop most distant from IFN interaction with its dimeric receptor. The biological activity for the PEG10-5(HGH)-IFN variant was determined to be 17% which is comparable to other PEGylated IFN conjugates achieved at or near the N-terminus that have been previously described. The lowest retained activity (10%) was reported for PEG10-120(HHH)-IFN which was prepared as a negative control targeting a IFN site thought to be involved in receptor binding. The presence of two histidines as a His2-tag to generate a site-selective target for bis-alkylating PEGylation is a feasible approach for achieving site-selective PEGylation. The use of a His2-tag to strategically engineer a conjugation site in a protein location can result in maximising the retention of the biological activity following protein modification.

Dataset Information

Site-Selective Cysteine-Cyclooctyne Conjugation.

Publications

Site-Selective Cysteine-Cyclooctyne Conjugation.

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