Structure of a PEGylated Protein Reveals a Highly Porous Double-Helical Assembly



Giada Cattani1, Lutz Vogeley2 and Peter B. Crowley1


  1. School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland.
  2. School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.


PEGylated proteins are a mainstay of the biopharmaceutical industry. Although the use of poly(ethylene glycol) (PEG) to increase particle size, stability and solubility is well-established, questions remain as to the structure of PEG–protein conjugates. Here we report the structural characterization of a model β-sheet protein (plastocyanin, 11.5 kDa) modified with a single PEG 5,000. An NMR spectroscopy study of the PEGylated conjugate indicated that the protein and PEG behaved as independent domains. A crystal structure revealed an extraordinary double-helical assembly of the conjugate, with the helices arranged orthogonally to yield a highly porous architecture. Electron density was not observed for the PEG chain, which indicates that it was disordered. The volume available per PEG chain in the crystal was within 10% of the calculated random coil volume. Together, these data support a minimal interaction between the protein and the synthetic polymer. Our work provides new possibilities for understanding this important class of protein–polymer hybrids and suggests a novel approach to engineering protein assemblies.

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Proteins were produced by transforming E. coli BL21 (DE3) with a modified pET11PC vector that encoded Pc Asp45Cys. Studler’s autoinduction method and purification was used to produce the protein except SEC was performed in a buffer with DTT to prevent disulfide formation. N-labelled protein, N-cysteine labelled protein and N-lysine-labelled proteins were all produced. A two step procedure in minimal medium (1 g 1-1 (NH4)SO4) was used to produce N-labelled protein. N-cysteine labelled protein was also produced in a minimal medium (50 mg 1-1 N-cysteine, 100 mg 1-1 all other amino acids). N-lysine labelled protein was produced in a similar minimal medium using 100 mg 1-1 N-Lys. These protein samples were analyzed in a mass spectrometer that had been modified with a CovalX HM2 detection system to gather mass data on PEG-Pc.


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