Amino acid-based hydrophobic affinity cryogel for protein purification from ora-pro-nobis (Pereskia aculeata Miller) leaves

Abstract

The surfaces of the polyacrylamide cryogels were coated with L-tryptophan (cryogel-Trp) or L-phenylalanine (cryogel-Phe) to enhance crude leaf extract-derived ora-pro-nobis (OPN) protein binding via pseudo-specific hydrophobic interactions. Cryogels functionalized with amino acids were prepared and characterized through morphological, hydrodynamic, and thermal analyses. The adsorption capacities of cryogel-Phe and cryogel-Trp were evaluated in terms of type (sodium sulfate or sodium phosphate) and concentration (0.02 or 0.10 mol∙L−1) of saline solution, pH (4.0, 5.5, or 7.0), and NaCl concentration (0.0 or 0.5 mol∙L−1). The cryogel-Phe presented a higher adsorptive capacity, achieving its maximum value (q = 92.53 mg∙g−1) when the crude OPN crude leaf extract was diluted in sodium sulfate 0.02 mol∙L−1 + NaCl 0.50 mol∙L−1, at pH = 7.0. The dilution rate significantly (p < 0.05) affected the recovered protein amount after the adsorption and elution processes, reaching 94.45% when the feedstock solution was prepared with a crude extract 5 times. The zeta potential for the eluted OPN proteins was 5.76 mV (pH = 3.23) for both dilution rates. The secondary structure composition mainly included β-sheets (46.50%) and α-helices (13.93%). The cryogel-Phe exhibited interconnected pores ranging 20–300 μm in size, with a Young modulus of 1.51 MPa, and thermal degradation started at 230 °C. These results indicate that the cryogel-Phe exhibited satisfactory properties as promising chromatography support for use in high-throughput purification of crude leaf extract-derived OPN proteins.