Site-selective protein conjugation at histidine

Chemical Science
Karolina PeciakSteve Brocchini

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 depend...Continue Reading

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Citations

Apr 16, 2019·Chembiochem : a European Journal of Chemical Biology·Michael R MortensenKurt V Gothelf
May 2, 2020·Organic & Biomolecular Chemistry·Shinichi SatoHiroyuki Nakamura
Jan 24, 2019·Organic & Biomolecular Chemistry·Michael Rosholm MortensenKurt Vesterager Gothelf
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Aug 22, 2021·Biotechnology Advances·Hèctor López-LagunaUgutz Unzueta
Jan 30, 2021·Protein and Peptide Letters·Arnab ChowdhuryAnupam Bandyopadhyay
Nov 18, 2021·Journal of the American Chemical Society·Katsuya MaruyamaMotomu Kanai

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Methods Mentioned

BETA
affinity purification
acylation
glycosylation
ion exchange chromatography
ion exchange purification
IEC
protein assay

Software Mentioned

SHuffle®

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