*3.6. Purification and Refolding of FR*α

All steps performed for purification and refolding of FRα were carried out according to our previous report [41]. However, some different reagents were used. For cell body washing, we used 4 M urea instead of Triton X-114. The inclusion bodies were solubilized and purified with 8 M urea instead of 6 M guanidine HCl. Purification and refolding data are shown in Figure S5, and Tables S2 and S3.

#### *3.7. BLI Measurements*

The binding affinity of refolded FRα toward folate and folate conjugated peptide aptamers was measured by biolayer interferometry at 25 ◦C using a BLItz system (ForteBio) with kinetics buffer [10 mM PBS, pH 7.4, 0.5% (*w*/*v*) BSA and 0.01% (*v*/*v*) Tween 20]. The measurement procedure has been reported previously [41]. Streptavidin-coated biosensors (SA sensors were hydrolyzed for 2 h in 250 µL kinetics buffer and then soaked with 250 µL folate-PEG8-biotin (2.5 µM), or a variety of concentrations of 250 µL folate-peptide-PEG24-biotin conjugates at a stirring speed of 2200 rpm. Two baselines were measured for each sensor in kinetics buffer for 30 and 300 s prior to the immobilization and association step, respectively. Folate-PEG8-biotin or folate-peptide-PEG24-biotin conjugates immobilized to SA biosensors were dipped into FRα solutions for the association step. Dissociation was monitored in 250 µL immune assay kinetics buffer. To eliminate errors from non-specific binding of the analyte (FRα) on the SA biosensor chips, reference data with the same concentrations of analyte were also measured.

The obtained binding data were analyzed using a 1:1 local analysis mode applied with association and dissociation step corrections by the BLItz Pro1.2 software (ForteBio). The reference measurements were subtracted during data analysis to determine *k*a, *k*<sup>d</sup> and *K*D.

#### **4. Conclusions**

By conjugation with peptides the affinities of folate to the receptor were enhanced. The conjugation with designed peptides will be useful for enhancement of ligands affinities through the increase of binding sites.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/9/2152/ s1.

**Author Contributions:** Y.I., H.M., D.K. and M.Y. conceived the project. A.N., M.U. and K.K. synthesized the organic compounds. H.M. designed the peptide sequences in the conjugates. R.D. and H.M. constructed the protein overexpression systems and purified the proteins. R.D. and H.M. carried out the click reactions and the subsequent HPLC assays. R.D. and H.M. measured the binding kinetics. R.D., H.M., M.U. and Y.I. wrote the manuscript.

**Funding:** This project was funded by the Incentive Research Program of RIKEN (FY2016, H.M.) and JSPS-Turkey (I2016652, Y.I.). R.D. and A.N. were supported by the International Program Associate of RIKEN (IPA number: 151022) and SPDR (Grant number: 201801061156), respectively.

**Acknowledgments:** We thank the Support Unit for Bio-material Analysis, RIKEN CBS Research Resources Center for peptide synthesis, *N*-terminal modification with biotin-PEG<sup>24</sup> and HPLC purification. We thank Kenji Suzuki to help the computational design of the compounds. We are thankful to Katsunori Tanaka, Masashi Ueki and Yun Heo for their input in CuAAC reaction. We are also thankful to Primetech Corp. for help with analysis of the BLI data. We thank the Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
