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The bifunctional linker-protein G (LPG) fusion protein comprises a peptide (linker) sequence and a truncated form of Streptococcus strain G148 protein G (protein G). The linker represents a multimeric solid-binding peptide (SBP) comprising 4 × 21-amino acid sequence repeats that display high binding affinity towards silica-based materials. In this study, several truncated derivatives were investigated to determine the effect of the SBP oligomerization on the silica binding function of LPG (for the sake of clarity, LPG will be referred from here on as 4 × LPG). Various biophysical characterization techniques were used to quantify and compare the truncated derivatives against 4 × LPG and protein G without linker (PG). The derivative containing two sequence repeats (2 × LPG) showed minimal binding to silica, while the truncated derivative with only a single sequence (1 × LPG) displayed no binding. The derivative containing three sequence repeats (3 × LPG) was able to bind to silica with a binding affinity of KD = 53.23 ± 4.5 nM, which is 1.5 times lower than that obtained for 4 × LPG under similar experimental conditions. Circular dichroism (CD) spectroscopy and fluorescence spectroscopy studies indicated that the SBP degree of oligomerization has only a small effect on the secondary structure (the linker unravels the beginning of the protein G sequence) and chemical stability of the parent protein G. However, based on quartz crystal microbalance with dissipation monitoring (QCM-D), oligomerization is an important parameter for a strong and stable binding to silica. The replacement of three sequence repeats by a (GGGGS)12 glycine-rich spacer indicated that the overall length rather than the SBP oligomerization mediated the effective binding to silica.
Bibliographical noteCopyright the Author(s) 2020. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.
- Linker-protein G
- Quartz crystal microbalance with dissipation monitoring (QCM-D)
- Silica-based materials
- Silica-binding peptide
- Solid-binding peptide (SBP)