Issue 2, 2019

Site-selective protein conjugation at histidine

Abstract

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.

Graphical abstract: Site-selective protein conjugation at histidine

Supplementary files

Article information

Article type
Edge Article
Submitted
28 Jul 2018
Accepted
09 Oct 2018
First published
09 Oct 2018
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2019,10, 427-439

Site-selective protein conjugation at histidine

K. Peciak, E. Laurine, R. Tommasi, J. Choi and S. Brocchini, Chem. Sci., 2019, 10, 427 DOI: 10.1039/C8SC03355B

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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