Human serum albumin specific recognition polypeptide and its application
A compound and conjugate technology, applied in the field of human serum albumin specific recognition of peptides, can solve problems such as the comparison of affinity and specific interaction between peptides and proteins
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[0055] Example Polypeptides that specifically bind to human serum albumin
[0056] 1) Characterization of the interaction between single-branched linear peptides and HSA
[0057] The single-branched peptides 1, 2, and 3 modified with cysteamine (with sulfhydryl groups) were dissolved in pH=7.40 phosphate buffer (150 mM PBS) to prepare a polypeptide solution with a concentration of 0.0013 mol / L. The solution was dropped on the surface of the SPRi chip, each peptide was dropped into three sample spots in parallel, incubated at 4°C for 12 h, the peptides were immobilized on the surface of the SPRi chip by Au-S bonds, and the unreacted peptide solution was washed with PBS and water, and the chip was put into Incubate in 5% (m / v) milk / PBS solution at 4°C for 12 h to block unreacted sites on the bare chip. Chips were washed again with PBS and water before use.
[0058] The data acquisition of SPRi was carried out using the Plexarray HT system. The flow rate of the solution was 2 μ...
example 2 7
[0068] Example 2, 7 High selectivity to HSA
[0069] The interaction between 7 and biomolecules was characterized using the SPRi chip modified with the heterobranched peptide 7 in Example 1 and 3). The solution, flow rate, acquisition of baseline, dissociation and elution conditions of the system are the same as 3) in Example 1. 3.76 μM of glucose, glutathione, deoxyribonucleoside, papain, pepsin, and bovine serum albumin (BSA) were introduced on the surface of the 7-functionalized chip. Its SPRi sensing signal such as Figure 5. It can be seen that 7 has good selectivity to HSA. It is worth mentioning that BSA and HSA are 76% similar in structure, such as Image 6 a, 6b, but 7 still has good selectivity to HSA. The analysis shows that for target 2, HSA and BSA are QDSISS and QDTISS, respectively. Although the two peptides have strong similarity in sequence and structure, the crystal structure shows that target 2 of HSA is stretched, while BSA is helical, and the stretche...
example 3 7
[0070] Example 3.7 Half-effect inhibitory activity of anti-HSA-FITC
[0071] A 96-well plate was coated with 10 μg / mL HSA carbonate solution (0.1 M, pH=9.6), incubated at 4°C for 12 h, and the washing steps were the same as those in Example 1 and 1). 7 was added to the HSA-coated 96-well plate at 100 μL / well of phosphate buffered saline (PBS) with a concentration ranging from 5 nM to 100 μM, and three groups of each concentration were incubated at 37° C. for 2 h and washed. Then, 5 μg / mL of anti-HSA-FITC phosphate buffer was added to the wells after 7 incubation, incubated at 37°C for 1 h in the dark, washed, set the excitation wavelength to 490 nm and the emission wavelength to 525 nm, and read with SpectraMax M5 Microplate reader. fluorescence value. like Figure 7 , and the ordinate is the ratio of the fluorescence intensity of each well to the fluorescence intensity of anti-HSA-FITC combined with HSA (I / I 0 ). When the fluorescence intensity of anti-HSA-FITC decreased ...
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