Giada Cattani1, Lutz Vogeley2 and Peter B. Crowley1
- School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland.
- 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.
CovalX Technology Used (Click each option to learn more)
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.