We chose sfGFP as a small model protein to investigate the competition to binding of FcRn to serum albumin and the serum half-life extension upon fatty acid conjugation. Although green fluorescent protein is not a therapeutic protein, it has been widely used in biomedical applications [40
]. In particular, its unique spectral properties including fluorescence and biocompatibility, make sfGFP a great surrogate for a therapeutic protein in drug delivery studies [41
]. In order to investigate the protein-size dependency of serum half-life extension via fatty acid conjugation, a model protein should be much smaller than Uox (140 kDa). Considering that the molecular weight of sfGFP with an anti-hexahistidine (6×His) tag used in this study is about 28 kDa, sfGFP was suitable as a small model protein. Purified sfGFP was prepared as described previously [8
]. Briefly, the gene of sfGFP was overexpressed in TOP 10 E. coli
cells. Then, the recombinant sfGFP was purified via metal affinity chromatography using its 6×His tag. The band of purified sfGFP was observed in the Coomassie blue–stained protein gel (Figure 2
B, lane 1), indicating that the purity of sfGFP was greater than 95%.
Next, four linkers with various lengths (LK01, 02, 03, and 04) were conjugated to sfGFP to generate sfGFP-PA conjugates (SP01, 02, 03, and 04) (Figure 2
A). The same four linkers were previously used to prepare Uox-PA conjugates [26
]. In general, properties of the fatty acid linker, such as solubility and length, may affect serum albumin binding affinity. However, for Uox-PA conjugates, the linkers did not exhibit substantial differences in serum albumin binding affinity [26
]. Therefore, in order to minimize the effects of fatty acid linkers on serum albumin binding, we chose the same set of linkers for sfGFP-PA conjugates. The lengths of linkers were estimated as 0.25, 1.5, 2.8, and 4.8 nm, respectively [26
]. Since these linker lengths were estimated at a maximal stretch, the actual linker lengths could be shorter than those theoretical values. The four sfGFP-PA conjugates (SP01, 02, 03, and 04) were prepared similarly to those of Uox-PA [26
]. Briefly, for SP01, NHS-PA (Figure S1a in Supplementary Materials
) was directly conjugated to lysine residues of sfGFP via NHS-amine reaction (Figure S1b in Supplementary Materials
). In the case of SP02, azidoacetic acid NHS ester (Figure S1c in Supplementary Materials
) was reacted with lysine residues of sfGFP to generate sfGFP-azides. DBCO-amine (Figure S1d in Supplementary Materials
) was reacted with NHS-PA to generate an intermediate (DBCO-PA) (Figure S1e in Supplementary Materials
). Then, DBCO-PA was reacted with sfGFP-azides via strain-promoted alkyne-azide cycloaddition (SPAAC) reactions (Figure S1f in Supplementary Materials
) to obtain SP02. In the case of SP03, azido-PEG4-NHS (Figure S1g in Supplementary Materials
) was reacted with lysine residues of sfGFP to generate sfGFP-PEG4-azides. Then, DBCO-PA was reacted with sfGFP-PEG4-azides to generate SP03. Finally, for SP04, NHS-PA was reacted with DBCO-PEG4-amine (Figure S1h in Supplementary Materials
) to generate DBCO-PEG4-PA (Figure S1i in Supplementary Materials
). Then, DBCO-PEG4-PA was reacted with sfGFP-PEG4-azides to generate SP04.
PA conjugation to the four sfGFP-PA conjugates was verified by protein band shifts in the protein gel and mass shifts in MALDI-TOF mass spectra (Figure 2
B and Figure S2 in Supplementary Materials
). In the protein gel, the bands of sfGFP-PA conjugates (SP01, 02, 03, and 04) were shifted up from the band of unmodified sfGFP, indicating the mass of sfGFP increased upon PA conjugation. Furthermore, we performed a MALDI-TOF spectrometric analysis of sfGFP and sfGFP-PA conjugates. The mass of unmodified sfGFP determined by MALDI-TOF analysis was 27,705 Da, which was consistent with its theoretical value (27,604 Da), with a 0.37% error. For sfGFP-PA conjugates, peaks were right-shifted, indicating that the mass of sfGFP increased upon PA conjugation. For all four sfGFP-PA conjugates, the mass differences between sfGFP-PA conjugate and unmodified sfGFP indicated that the average number of PA conjugated to single sfGFP was about one. Therefore, we speculated that the property differences among sfGFP-PA conjugates resulting from the different number of conjugated PAs were minimal.